Interferon-γ inducing polypeptide, pharmaceutical composition thereof, monoclonal antibody thereto, and methods of use

ABSTRACT

A human IFN-γ inducing polypeptide and its nucleotide sequence was isolated, purified and characterized. Pharmaceutical compositions containing this novel INF-γ inducing polypeptide or active fragments thereof are formulated and monoclonal antibodies are raised against this polypeptide or antigenic fragments thereof. The polypeptide can be used in a method for treating diseases susceptive to treatment with INF-γ, and methods for enhancing the cytotoxicity of NK cells or for inducing the formation of LAK cells, such as to treat tumors and malignant pathologies.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of application Ser. No. 09/716,356,filed Nov. 21, 2000, which is a continuation-in-part of: applicationSer. No. 08/832,180, filed Apr. 8, 1997, which is a divisional of Ser.No. 08/558,191, filed Nov. 15, 1995, now abandoned; application Ser. No.08/974,469, filed Nov. 20, 1997, which is a continuation of Ser. No.08/599,879, filed Feb. 14, 1996, now abandoned, which application Ser.No. 08/599,879 is a continuation-in-part of Ser. No. 08/558,190, filedNov. 15, 1995, now abandoned; application Ser. No. 08/558,818, filedNov. 15, 1995; application Ser. No. 08/832,177, filed Apr. 8, 1997,which is a divisional of Ser. No. 08/558,818; and application Ser. No.08/832,198, filed Apr. 8, 1997, which is a divisional of Ser. No.08/721,018, filed Sep. 26, 1996, now abandoned. Each of theabove-identified applications for which the present application is acontinuation-in-part is incorporated entirely herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a polypeptide and fragments thereofwhich induce interferon-γ (INF-γ) production by immunocompetent cells, apharmaceutical composition containing same, and a monoclonal antibodyspecific for this polypeptide. The present invention also relates to aDNA encoding the INF-γ production inducing polypeptide or peptide,methods of using the polypeptide or fragments thereof, pharmaceuticalcompositions, or monoclonal antibodies.

2. Description of the Related Art

Interferon-γ (INF-γ) is a protein which is known to have antiviral,antioncotic, and immunoregulatory activities, and which is produced byimmunocompetent cells stimulated with antigens or mitogens. Because ofthese biological activities, INF-γ was expected to be used as anantitumor agent and was tested in clinical trials as a therapeutic fortreating malignant tumors in general, including brain tumors. INF-γpreparations, which are now commercially available, are roughlyclassified into two groups, natural INF-γ polypeptides produced byimmunocompetent cells and recombinant INF-γ polypeptides produced inEscherichia coli transformed with a DNA which encodes for natural INF-γ.In the clinical trials, either natural INF-γ polypeptide or recombinantINF-γ is administered to patients as an exogenous INF-γ.

Natural INF-γ polypeptides are usually produced by culturing establishedimmunocompetent cells in nutrient culture media supplemented with INF-γinducers to produce INF-γ polypeptides, and then purifying the producedINF-γ polypeptides. It is known that the type of INF-γ inducer used inthe nutrient culture media greatly influences the production yield ofINF-γ polypeptide as well as the ease of INF-γ purification and thesafety of the final INF-γ preparations. Generally, mitogens such asconcanavalin A (Con A), lentil lectin from Lens culinaris, pokeweedpectin from Phytolacca americana, endotoxin and lipopolysaccharide canbe used as INF-γ inducers. However, these mitogens have problems withthe molecular variety and quality of the preparation, which depend onthe origin of the mitogen and purification methods used, as well as onproduction of mitogens with constant INF-γ inducibility in satisfactoryyields. In addition, most of these mitogens induce unfavorable sideeffects when administered in vivo, with some even showing toxicity. As aresult, it is not practical to use such mitogens to induce INF-γproduction by direct in vivo administration to a patient.

Recently, some pharmaceuticals which contain as an effective ingredienta cytokine, such as interferon-α, interferon-β, TNF-α, TNF-β,interleukin 2, and interleukin 12, as well as INF-γ, were developed orare being explored for actual use. These pharmaceuticals can be used asan antitumor agent, antiviral agent, antiseptic or immunoregulatoryagent and, if necessary, they can be used together with othermedicaments.

Unlike chemically-synthesized pharmaceuticals, the aforesaidpharmaceuticals have the characteristic that they can be administered topatients for a relatively long period of time without inducing seriousside effects. However, they also have the drawback that theirtherapeutic effects are relatively low, and they cannot substantiallyabate or cure diseases when used alone; the results vary depending onthe types of diseases and symptoms to be treated. Accordingly, thesepharmaceuticals are now used as a supplement to chemically-synthesizedagents in the treatment of serious diseases, such as malignant tumors,to prolong the patient's life.

SUMMARY OF THE INVENTION

The present invention provides an INF-γ inducing factor which is apurified polypeptide that induces INF-γ production by immunocompetenthuman cells. As an embodiment of the purified factor, a purifiedpolypeptide which induces INF-γ production by immunocompetent humancells and is obtainable from human cells is also provided.

The present invention further provides a pharmaceutical compositioncontaining the INF-γ inducing polypeptide, where the pharmaceuticalcomposition may advantageously include either interleukin-2 orinterleukin-12.

An aspect of the present invention relates to methods of using the INF-γinducing polypeptide. One embodiment is directed to a method fortreating atopic diseases, tumors, viral diseases, bacterial diseases, orimmunopathies. In particular, a method for enhancing the cytotoxicity ofNK cells and method for inducing the formation of LAK cells areprovided.

Also provided by the present invention is a DNA molecule encoding theINF-γ inducing polypeptide according to the present invention, areplicable recombinant DNA containing a self-replicable vector and theDNA molecule encoding the INF-γ inducing polypeptide, host cellstransformed with the self-replicable vector, and a process for preparingthe INF-γ inducing polypeptide.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an HPLC elution pattern profile of peptide fragments obtainedby trypsinizing a protein derived from mouse liver.

FIG. 2 is a schematic representation of the structure of recombinantplasmid pHIGIF, where HIGIF cDNA represents the cDNA encoding the INF-γinducing polypeptide, Ptac represents the tac promoter, GST representsglutathione S transferase gene, AmpR represents ampicillin resistancegene, and ori represents an Escherichia coli replication initiation sitepresent on plasmid pBR322.

FIG. 3 is an HPLC elution pattern/profile of peptide fragments obtainedby peptide mapping an INF-γ inducing protein of human cell origin usingclostripain.

FIG. 4 is a schematic representation of the structure of recombinantplasmid pKGFHH2, where KGFHH2 cDNA represents the cDNA encoding theINF-γ inducing polypeptide, Ptac represents the tac promoter, rrnBT1T2represents a terminator of a ribosomal RNA operon, AmpR representsampicillin resistance gene, and pBR322 ori represents an Escherichiacoli replication initiation site.

FIG. 5 is an image of a Western blot showing the reactivity of thepresent purified polypeptide and human interleukin 12 with themonoclonal antibody H-1mAb.

DETAILED DESCRIPTION OF THE INVENTION

Polypeptide/Protein and Encoding DNA

The present invention is based on the initial discovery of a substancein mouse liver which induces INF-γ production during studies ofcytokines produced from mammalian cells in the laboratories of thepresent inventors. The present inventors isolated this substance byusing a variety of purification methods including column chromatographyas a main technique, and studied the properties and features of thissubstance, revealing that it is a protein with the followingphysicochemical properties:

-   -   (1) Molecular Weight Exhibiting a molecular weight of        19,000±5,000 daltons on sodium dodecyl polyacrylamide gel        electrophoresis (SDS-PAGE);    -   (2) Isoelectric Point (pI) Exhibiting an isoelectric point of        4.8±1.0 on chromatofocusing;    -   (3) Partial Amino Acid Sequence Having the partial amino acid        sequences in SEQ ID NOs: 1 and 2; and    -   (4) Biological Activity Inducing the INF-γ production by        immunocompetent cells.

This substance is a novel protein whose physicochemical properties havenot been previously reported.

Based on the partial amino acid sequences obtained above, primers werechemically synthesized and used with MRNA isolated from mouse livercells as template in a reverse transcription-polymerase chain reaction(RT-PCR) to generate DNA fragments which partially encode the protein.By using the generated DNA fragments as probes, a cDNA library preparedfrom MRNA was screened and it was found that the DNA of the substanceisolated from mouse liver cells consists of 471 base pairs (SEQ ID NO:3)and encodes the 157 residue amino acid sequence of SEQ ID NO:4, whereXaa represents methionine or threonine.

The present inventors further studied mRNA derived from human livercells, and found a human gene that encodes a polypeptide which inducesINF-γ production by immunocompetent cells. This gene contains thenucleotide sequence of SEQ ID NO:5 and encodes a 157 amino acid residuepolypeptide of SEQ ID NO:6, where Xaa represents isoleucine orthreonine.

The steps and techniques used to obtain the nucleotide and amino acidsequences of SEQ ID NOs:5 and 6 are summarized below.

(1) A protein, which induces INF-γ production by immunocompetent cells,was isolated from mouse liver cells and was highly purified by combiningconventional purification methods that included chromatography as a maintechnique.

(2) The resultant highly purified protein was digested with trypsin, andtwo polypeptide fragments were isolated from the resultant mixture andanalyzed to determine their amino acid sequences.

(3) From mouse liver cells, mRNA was collected and used as a template ina reverse transcription-polymerase chain reaction (RT-PCR) witholigonucleotide primers (chemically synthesized based on the abovepartial amino acid sequences obtained from the polypeptide fragments).The DNA fragments were then screened with oligonucleotide probes whichhad been chemically synthesized based on partial amino acid sequences,followed by collecting a DNA fragment which partially encodes theprotein.

(4) The resultant DNA fragments were labeled and hybridized with a cDNAprepared, from using mouse liver mRNA as template, followed by selectionof a transformant which exhibited strong hybridization.

(5) A cDNA was isolated from the transformant, and the nucleotidesequence was determined. Comparison of the deduced amino acid sequenceand the partial amino acid sequence obtained earlier revealed that theprotein has the amino acid sequence of SEQ ID NO:4. In mice, thenucleotide sequence of SEQ ID NO:3 encodes this amino acid sequence ofSEQ ID NO:4.

(6) A DNA fragment having the nucleotide sequence of SEQ ID NO:3 wasprepared, labeled and hybridized to a cDNA library which had beenprepared from mRNA derived from human liver cells as templates, followedby selecting a transformant which exhibited strong hybridization to SEQID NO:3.

(7) The cDNA prepared from the transformant was sequenced. The proteinis a polypeptide which comprises the amino acid sequence of SEQ ID NO:6as encoded by the nucleotide sequence of SEQ ID NO:5 in humans.

Through long term research, the present inventors have found the presentpolypeptide which induces INF-γ production by immunocompetent cells whenallowed to act alone or together with an appropriate cofactor. As isevident from SEQ ID NO:6, this novel polypeptide, which has a molecularweight of about 18,500±3,000 on sodium dodecyl sulfate polyacrylamidegel electrophoresis (SDS-PAGE) and an isoelectric point of about 4.9±1.0on chromatofocusing, differs from conventionally known polypeptides. Thepresent polypeptide includes natural and recombinant polypeptides aslong as they have the amino sequence of SEQ ID NO:6, where Xaa standsfor isoleucine or threonine, or sequences homologous to SEQ ID NO:6.Variants, which have amino acid sequences homologous to SEQ ID NO:6, canbe obtained by replacing one or more amino acid residues in SEQ ID NO:6with different amino acid residues, by adding one or more amino acidresidues to the N- and/or C-termini of SEQ ID NO:6, or by deleting oneor more amino acid residues at the N- and/or C-termini of SEQ ID NO:6without altering the inherent biological activity of the present INF-γinducing polypeptide. Depending on the host cells into which the DNAsare introduced (even when the same DNAs are used) and on the componentsand the conditions of cultivation such as temperature and pH fortransformant containing the DNA, variants may be generated which eitherlack one or more amino acid residues at the N- and/or C-termini in SEQID NO:6, such as fragments of SEQ ID NO:6, or contain one or moreadditional amino acid residues near the N-terminus of SEQ ID NO:6through modification by internal enzymes of the host cell following DNAexpression, while retaining the inherent biological properties of thepolypeptide. The polypeptide is intended to encompass such variants aslong as the variants induce INF-γ production by immunocompetent cells.

The present interferon-γ inducing polypeptide can be prepared byculturing transformants, which contain the encoding DNA for thepolypeptide to produce the interferon-γ inducing polypeptide, andcollecting the polypeptide from the resultant cultures. Thetransformants usable in the present invention can be obtained by, forexample, introducing DNA having the nucleotide sequence of SEQ ID NO:5,sequences homologous thereto, or complementary sequences into hostcells. One or more nucleotides in the nucleotide sequences can bereplaced with different nucleotides by means of the degeneracy of thegenetic code without altering the amino acid sequence of the presentpolypeptide. To express and produce the present polypeptide in hostcells using such DNAs, one or more nucleotides in the nucleotidesequences which encode the present polypeptide or its variants can bereplaced with different nucleotides.

Any DNA encoding the INF-γ inducing polypeptide, i.e., produced fromnatural sources or produced artificially, can be used in the presentinvention independent of their origin. Natural sources include, forexample, human liver cells from which the gene containing the DNA withnucleotide sequence of SEQ ID NO:7 is obtainable. The preparationprocedure is as follows: 1) fractionating to isolate poly(A)⁺ RNA from acommercially available human liver RNA on a sucrose gradient buffer; 2)allowing a reverse transcriptase and a polymerase to act on the mRNA asa template to generate double-stranded cDNA; 3) introducing the cDNAgenerated into an appropriate self-replicable vector, and transformingan appropriate host such as Escherichia coli with the resultantrecombinant DNA; 4) culturing the resultant transformant in a nutrientculture medium; and 5) collecting the proliferated transformantscontaining the DNA encoding the present polypeptide by the colonyhybridization method. The DNA according to the present invention isobtainable by treating the transformants with conventional methods. Toartificially produce the present DNA, for example, the DNA is preparedby chemical synthesis based on the nucleotide sequence of SEQ ID NO:5,or by introducing a DNA which encodes the amino acid sequence of SEQ IDNO:6 into an appropriate vector to form a recombinant DNA, introducingthe recombinant DNA into an appropriate host, culturing the resultanttransformant in a nutrient culture medium, isolating the proliferatedtransformed host cells from the culture, and collecting and recoveringplasmids containing the objective DNA from the cells.

Generally, the DNA was introduced into host cells in the form of arecombinant DNA. Such a recombinant DNA usually contains the DNA and aself-replicable vector, and it can be readily prepared by recombinantDNA technology in general. Non-limiting examples of a suitableself-replicable vector are plasmid vectors such as pKK223-2, pGEX-2T,PRL-λ, pBTrp2 DNA, pUB110, Yep13, Ti plasmid, Ri plasmid and pBI121.From among these vectors, pKK223-2, pGEX-2T, PRL-λ, pBTrp2 DNA, pUB110and YEp13 are suitably used when the present DNA is to be transformedinto and expressed in yeast and procaryotes such as microorganisms ofthe species Escherichia coli and Bacillus subtilis, whereas Ti plasmid,Ri plasmid and pBI121 are suitably used when transformation andexpression in animal and plant cells is desired.

To incorporate the present DNA into these vectors, conventional methodsused in the field can be arbitrarily used: genes containing the presentDNA and self-replicable vectors are cleaved with restriction enzymesand/or ultrasonic treatment, and the resultant DNA fragments and vectorfragments are ligated. To cleave genes and vectors, restriction enzymes,which specifically act on nucleotide sequences, more particularly thosetype II restriction enzymes such as Sau3AI, EcoRI, HindIII, BamHI, SalI,XbaI, SacI and PstI, are used to facilitate the ligation of DNAfragments and vector fragments. To ligate the DNA fragments and vectorfragments, the fragments to be ligated are first annealed, if necessary,and then treated with a DNA ligase in vivo or in vitro. The recombinantDNAs thus obtained can be readily introduced into appropriate hostcells, and this would enable the unlimited replication of the DNAsthrough the culturing of transformed host cells.

The recombinant DNAs usable in the present invention can be introducedinto appropriate host cells such as cells of yeasts and microorganismsof the species Escherichia coli and Bacillus subtilis. Whenmicroorganisms of Escherichia coli are used as host cells, they arecultured in the presence of the recombinant DNAs and calcium ions, andwhen microorganisms of the species Bacillus subtilis are used as hostcells, the competent cell method and the protoplast method are used forobtaining transformants. In order to obtain the desired transformantclone, transformants are screened/selected by the colony hybridizationmethod or by culturing all the transformants in nutrient culture media,and screening/selecting those which produce polypeptides capable ofinducing INF-γ production by immunocompetent cells.

The transformants thus obtained produce the present polypeptideintracellularly or extracelluarly when cultured in nutrient culturemedia. Non-limiting examples of such nutrient culture media are liquidculture media which contain carbon sources, nitrogen sources andminerals, as well as amino acids and/or vitamins as a micronutrient. Thecarbon sources usable in the present invention include saccharides suchas starch, starch hydrolysates, glucose, fructose and sucrose. Thenitrogen sources usable in the present invention includenitrogen-containing organic and inorganic compounds such as ammonia andtheir salts, urea, nitrates, peptone, yeast extract, defatted soy bean,corn steep liquor, beef extract, etc.

Transformants are inoculated into nutrient culture media and incubatedat a temperature of 25-65° C. and at a pH of 5-8 for about 1-10 daysunder aerobic conditions by the agitation-aeration method, etc., toobtain cultures containing the present polypeptide. Although thecultures can be used intact as an INF-γ inducer, they are subjected, ifnecessary, to ultrasonication and/or cell lysis enzymes in order todisrupt cells, followed by filtering or centrifuging the resultantsuspensions to remove intact cells and cell debris, and further bypurifying the resultant supernatants containing the present polypeptide.The purification methods usable in the present invention are, forexample, those which are generally used in this field to purifybiologically active substances, i.e., concentration, salting out,dialysis, separatory sedimentation, gel filtration chromatography, gelelectrophoresis, and isoelectrophoresis, and, if necessary, two or moreof the purification methods can be used in combination. The resultantpurified solutions containing the present polypeptide can beconcentrated and/or lyophilized into liquids or solids suitable to meetthe final use of the polypeptide.

The polypeptide of the present invention can also be isolated from humancells as a protein. The term “polypeptide” as used herein for the humanINF-γ inducing polypeptide is intended to mean polypeptides andglycoproteins in general which induce INF-γ production byimmunocompetent cells and contain the amino acid sequence of SEQ IDNO:14. Depending on the type and conditions of propagating human cells,the polypeptide has the amino acid sequences of SEQ ID NOs:14 and 15 atthe N- or C-terminal region, respectively, and occasionally has theamino acid sequence of SEQ ID NO:6 (where Xaa represents isoleucine orthreonine) as a complete amino acid sequence, including the amino acidsequences of SEQ ID NOs:16 and 17 as an internal fragment. Whensubjected to peptide mapping using clostripain, peptide fragments of SEQID NOs:15, 16, 17 and 19 were observed. The polypeptide is detected as aprotein band corresponding to a molecular weight of 14,000-24,000daltons (usually 18,000-19,500 daltons) when determined on SDS-PAGE inthe presence of a reducing agent such as dithiothreitol. Depending onthe type and conditions of propagating human cells, one or more aminoacid residues may be added to the N- and/or C-termini (SEQ ID NOs:14 and15) or one or more amino acid residues at the N- or C-termini may bedeleted. Any INF-γ inducing polypeptide can be used in the presentinvention as long as it is derived from a human cell, and has either ofthese amino acid sequences and the activity of inducing INF-γ productionwhen acting on immunocompetent cells alone or together with anappropriate cofactor.

These INF-γ inducing polypeptides according to the present invention canbe produced by the present process using human cells. Usually, the humancells used in the present invention include cell lines derived fromhuman hematopoietic cells such as lymphoblasts, lymphocytes, monoblasts,monocytes, myeloblasts, myelocytes, granulocytes and macrophages.Non-limiting examples of these cell lines are lymphomas and leukemiassuch as myelocytic leukemia, promyelocytic leukemia, adult T-cellleukemia, and hairy cell leukemia, and more specifically, HBL-38 cell,HL-60 cell (ATCC CCL240), K-562 cell (ATCC CCL243), KG-1 cell (ATCCCCL246), Mo cell (ATCC CRL8066), THP-1 cell (ATCC TIB202), U-937 cell(ATCC CRL1593) as reported by Jun MINOWADA in Cancer Review, 10:1-18(1988), and A-253 cell (ATCC HTB41), which is an epidermoid carcinoma ofthe human submaxillary gland. Mutants of these cell lines can also beused in the present invention. Because these cell lines readilyproliferate and produce large quantities of the present polypeptide,they can be advantageously used in the present invention. In particular,epidermoid carcinoma cell lines such as A-253, and human myelomonocyticcell lines such as HBL-38, HL-60, KG-1, THP-1, and U-937 cells haveextremely high productivity for the present polypeptide and are mostsuitable for use in the present invention.

In the present process, the above-mentioned human cells are firstallowed to propagate, with the present polypeptide then being collectedfrom the propagated cells. The method used to propagate these humancells according to the present invention is not specifically limited,and any conventional in vivo or in vitro propagation method can be used.The in vitro propagation method is intended to mean a method forpropagating cells using nutrient culture media, which comprisessuspending human cells in RPMI 1640 medium, MEM medium and DMEM medium,which are conventionally used in the art for propagating animal cells,supplemented with 0.3-30 w/v % of fetal bovine serum to give a celldensity of about 1×10⁵-1×10⁶ cells/ml, and culturing these cells at atemperature of 36-38° C., preferably at a temperature of about 37° C.and at a pH of 7-8, more preferably at a pH of 7.2-7.4, for about 1-7days where the culture media are replaced with fresh culture media.Afterwards, the propagated cells were separated from the culture toobtain the objective polypeptide. Depending on the type and conditionsfor culturing human cells, some cells can excrete the presentpolypeptide extracellularly while being cultured. When inducers, such asmitogens and/or INF-γs which induce the production of the presentpolypeptide by human cells are present in the culture media, most or allof the polypeptide is produced extracellularly. In this case, thepolypeptide can be collected from the culture supernatant.

The method for in vivo propagation of human cells in warm-bloodedanimals other than humans includes injecting antithymocyte antibodiesderived from rabbits into rodents, such as newborn mice, nude mice,rats, nude rats, guinea pigs, and hamsters, to suppress immunoreactionin the animals, injecting subcutaneously or intraperitoneally about1×10⁵-1×10⁸ of human cells per animal into the animals or placing thehuman cells in diffusion chambers embedded within or outside of the bodyof the animals while allowing the body fluid of the animals to circulatein the chambers, and feeding the animals by conventional methods forabout 2-10 weeks. During the feeding, the human cells propagate in thepresence of body fluid from the animals. The propagated human cells arecollected in the form of a tumor mass, ascites or cell suspension. Ifnecessary, the objective polypeptide is collected after suspending andwashing these human cells with an appropriate solvent. The in vivopropagation method has an advantage over the in vitro propagation methodin that the desired cells can be obtained in a shorter period of time,at a lower labor cost and in a sufficiently high yield. The in vivopropagation method is disclosed, for example, in Japanese PatentPublication No. 54,158/81.

To collect and recover the present polypeptide from the propagatedcells, these cells are disrupted by ultrasonic energy before or afterseparation from the culture, homogenizing, freezing and thawing, or bysoaking these cells in low osmotic solvents, and then collecting thepolypeptide from the resulting cell debris or from a mixture of celldebris and culture supernatant. To collect the present polypeptide fromthe cell debris or the mixture, the cell debris or the mixture can besubjected directly, or after incubation at about 37° C. for 1-24 hours,to the following conventional methods for purifying biologically activesubstances in the art: salting out, dialysis, filtration, concentration,separatory sedimentation, gel filtration chromatography, ion-exchangechromatography, hydrophobic chromatography, adsorption chromatography,affinity chromatography, chromatofocusing, gel electrophoresis and/orisoelectrophoresis. Two or more of these conventional methods can beselectively used in combination. The collected polypeptide can beconcentrated and/or lyophilized into a liquid or solid form for itsintended final use. The monoclonal antibody according to the presentinvention, as discussed below, is advantageously used to purify thepresent polypeptide. Immunoaffinity chromatography using the monoclonalantibody yields the highest possible purity of the present polypeptideat the lowest cost and effort.

Agent, Pharmaceutical Compositions and Methods of Use

As described in the preceding section, the polypeptide as an agentaccording to the present invention has a property of inducing stableINF-γ production by immunocompetent cells. INF-γs are well-known tocontribute to human biophylaxis through their protection againstinfectious bacteria, their growth inhibitory activity on malignanttumors, their immunoregulatory activity, and their inhibitory activityon the production of immunoglobulin E antibody.

In Cytokines in Cancer Therapy, edited by Frances R. Balkwill,translated by Yoshihiko WATANABE (1991), published byTokyo-Kagaku-Dojin, Tokyo, Japan, it is reported that almostsatisfactory results were obtained when a treatment, using killer cellssuch as natural killer (NK) cells and lymphokine-activated killer (LAK)cells, was applied on a variety of human diseases including antitumorimmunotherapy. Recently, it has been noted that there is a relationshipbetween the therapeutic effect and the induction of killer cells or theenhancement of the cytotoxicity by killer cells using cytokines. Forexample, T. FUJIOKA reported in British Journal of Urology, 73 (1):23-31 (1994), that in antitumor immunotherapy using LAK cells andinterleukin 2, interleukin 2 strongly induced LAK cell formation andexerted a remarkable cancer metastasis-inhibitory activity on humancancers without causing serious side effects. Thus, it is shown thatINF-γ and killer cells are closely involved in the treatment and/orprevention of a variety of human diseases, and greatly contribute totheir treatment or remission.

When administered to humans, the present agent induces INF-γ productionby immunocompetent cells in vivo, and exerts a satisfactory therapeuticand/or prophylactic effect on diseases susceptible to treatment withINF-γ, including viral diseases such as AIDS and condyloma acuminatum;malignant tumors such as malignant nephroma, granuloma, mycosisfungoides, and brain tumor; and immunopathies such as articularrheumatism and allergy. The use of this agent according to the presentinvention is not restricted to only humans, but can include othermammals such as mouse, rat, hamster, dog, cat, cow, horse, goat, sheep,pig, and monkey.

Because the present polypeptide induces INF-γ production by humanimmunocompetent cells, agents for diseases susceptible to treatment withINF-γ and containing the polypeptide as an effective ingredientstimulate human immunocompetent cells to produce INF-γ by administeringthe polypeptide to humans, and exert a positive effect on the treatmentand/or prevention of INF-γ susceptive diseases. The polypeptide havingthe amino acid sequence of SEQ ID NO:6 has the properties of enhancingthe cytotoxicity of killer cells, i.e., NK cells, LAK cells, cytotoxicT-cells, and inducing INF-γ production by immunocompetent cells withoutcausing serious side effects. Killer cells participate in the treatmentand/or the prevention of diseases susceptible to treatment with INF-γwhen the present polypeptide is used to induce INF-γ production byimmunocompetent cells in vivo, to enhance the cytotoxicity of killercells such as cytotoxic T-cells and NK- and LAK-cells, and to induce theformation of killer cells similar to the polypeptides described in thelater described Examples. When the INF-γ inducing polypeptide augmentsthe cytotoxicity of killer cells or induces the formation of killercells, it exerts a strong effect in treating inveterate diseases such asmalignant tumors. It can also be used together with interleukin 2 and/ortumor necrosis factor to improve the therapeutic effect and reduce theside effects in treatments using adoptive immunity for malignant tumorsincluding solid tumors such as lung cancer, renal cancer, and breastcancer.

Because the present polypeptide has a strong INF-γ productioninducibility and has relatively low toxicity, it can induce a desiredlevel (amount) of INF-γ production in a small amount. The presentpolypeptide does not cause serious side effects even when administeredto patients at a relatively high dose because it has low toxicity.Therefore, the present polypeptide is advantageous in that it quicklyinduces a desired level of INF-γ production without strictly controllingthe dose. The present polypeptide, when of human cell origin, isparticularly advantageous in that it causes less side effects andinduces less antibodies when administered to humans in the form of apharmaceutical composition as compared with polypeptides producedartificially by recombinant DNA techniques.

The term “diseases susceptible to treatment with INF-γ” as used hereinmeans diseases in general which can be directly or indirectly treatedand/or prevented by INF-γ and/or killer cells. For example, viraldiseases such as hepatitis, herpes syndrome, condyloma acuminatum, andAIDS; infectious diseases such as candidiasis, malaria, cryptococcosis,and Yersinia; malignant solid tumors such as renal cancer, mycosisfungoides, and chronic granulomatous disease; hematopoietic malignanttumors such as adult T-cell leukemia, chronic myelocytic leukemia, andmalignant leukemia; and immunopathies/immune diseases such as allergyand rheumatism. When used with interleukin 3, the present polypeptideexerts a strong effect on the treatment or the remission of leukopeniaand thrombocytopenia induced by radio- and chemotherapies for treatingleukemia, myeloma, and malignant tumors.

The present agent for susceptive diseases can be widely used in thetreatment and/or the prevention of the above diseases as an antitumoragent, antiviral agent, antiseptic, immunotherapeutic agent,platelet-increasing agent, or leukocyte-increasing agent. Depending onthe type of agents and the symptom of diseases to be treated, thepresent agent is generally processed into a liquid, paste or solid formwhich contains 0.000001-100 w/w %, preferably 0.0001-0.1 w/w % of thepresent polypeptide, on a dry solid basis (d.s.b.).

The present agent can be used intact or processed into compositions bymixing with physiologically-acceptable carriers, adjuvants, excipients,diluents and/or stabilizers, and, if necessary, further mixing with oneor more biologically-active substances such as interferon-α,interferon-β, interleukin 2, interleukin 3, interleukin 12, TNF-α,TNF-β, carboquone, cyclophosphamide, aclarubicin, thiotepa, busulfan,ancitabine, cytarabine, 5-fluorouracil, 5-fluoro-1-(tetrahydro-2-furyl)uracil, methotrexate, actinomycin D, chromomycin A₃, daunorubicin,doxorubicin, bleomycin, mitomycin C, vincristine, vinblastine,L-asparaginase, radio gold colloidal, Krestin®, picibanil, lentinan, andMaruyama vaccine. Among these combinations, a combination of the presentpolypeptide and interleukin 2 is especially useful because interleukin 2acts as a cofactor for the present polypeptide when the presentpolypeptide induces IFN-γ production by immunocompetent cells. Thecombination of the present polypeptide and a natural or recombinanthuman interleukin 2 induces a relatively high level of INF-γ productionusing only a small amount of the present polypeptide, which by itselfdoes not substantially induce INF-γ production by immunocompetent cells.A combination of the present polypeptide and interleukin 12 also inducesa greater level of INF-γ production than could be readily attained bythe present polypeptide alone. Because the present polypeptide increasesthe inhibitory activity of interleukin 12 on the production ofimmunoglobulin E antibody in the human body, the present polypeptide canbe advantageously used to treat immunopathies such as atopic diseasesincluding atopic asthma, atopic bronchial asthma, hay fever, allergicrhinitis, atopic dermatitis, angioedema, and atopic disorders of thedigestive system. The sole administration of the present polypeptide tohumans can attain the desired therapeutic effect because interleukin 12is inherently present in small amounts in the human body.

The form of the present agent for diseases susceptible to treatment withINF-γ includes those in a unit dose form, which are physicallyformulated medicaments suitable for administration and contain thepresent polypeptide in a daily dose or in a dose from 1/40to severaltimes (i.e., up to 4 time) the daily dose. Examples of these medicamentsare injections, liquids, powders, granules, tablets, capsules,sublinguals, opthalmic solutions, nasal drops, and suppositories.

The present agent can be orally or parenterally administered topatients, and as described below, it can be used to activate antitumorcells in vitro. In both administrations, the agent exerts a satisfactoryeffect in the treatment and/or the prevention of diseases susceptible totreatment with INF-γ. Although it varies depending on the types ofdiseases susceptible to treatment with INF-γ and the symptoms ofpatients before and after administration, the agent can be orallyadministered to patients or parenterally administered to the intradermaltissues, subcutaneous tissues, muscles, and veins of patients at a doseof about 0.1 μg to 50 mg per shot, preferably about 1 μg to 1 mg pershot, 1-4 times/day or 1-5 times/week, for one day to one year.

The present agent can be also used in a so-called “antitumorimmunotherapy” using interleukin 2. Generally, antitumor immunotherapyis roughly classified into (i) a method for directly administeringinterleukin 2 to patients with malignant tumors, and (ii) a method forintroducing cells which were previously activated in vitro byinterleukin 2, i.e., adoptive immunotherapy. The immunotherapeuticeffect can be significantly enhanced when the present polypeptide isadministered in combination with interleukin 2. In method (i), thepresent polypeptide is administered to patients in an amount of about0.1 μg/shot/adult to 1 mg/shot/adult either for 1-10 times before theadministration of interleukin 2 or simultaneously with interleukin 2.The dose of interleukin 2 is generally about 10,000-1,000,000units/shot/adult, although it varies depending on the types of malignanttumors, patients' symptoms, and the dose of the present polypeptide. Inmethod (ii), mononuclear cells and lymphocytes collected from patientswith malignant tumors are cultured in the presence of interleukin 2 andabout 0.1 ng to 1 μg of the polypeptide per 1×10⁶ blood cells. Afterculturing for a prescribed period of time, NK or LAK cells werecollected from the culture and introduced back into the same patient.Diseases which can be treated by the present antitumor immunotherapyare, for example, hematopoietic malignant tumors such as leukemia andmalignant lymphoma, solid malignant tumors such as colonic cancer,rectal cancer, large intestinal cancer, gastric cancer, thyroidcarcinoma, cancer of the tongue, bladder carcinoma, choriocarcinoma,hepatoma, prostatic cancer, carcinoma uteri, laryngeal, lung cancer,breast cancer, malignant melanoma, Kaposi's sarcoma, cerebral tumor,neuroblastoma, tumor of the ovary, testicular tumor, osteosarcoma,cancer of the pancreas, renal cancer, hypernephroma, andhemangioendothelioma.

The present polypeptide can also be used as an inducer for INF-γproduction by cell culture methods and is usually added to nutrientculture media for INF-γ production in cultured immunocompetent cells.Leukocytes separated from mammalian peripheral blood or established celllines of immunocompetent cells such as HBL-38 cell, Mo cell (ATCCCRL8066), Jurkat cell (ATCC CRL8163), HUT78 cell (ATCC TIB161), EL4 cell(ATCC TIB39), L-12-R4 cells, and mutants thereof are suspended inculture media containing about 0.1-1,000 ng/ml of the presentpolypeptide, preferably about 1-100 ng/ml of the present polypeptide. Ifnecessary, these cells are cultured in nutrient culture mediasupplemented with T-cell stimulants such as mitogens, interleukin 2, andanti-CD3 antibody for about 1-100 hours and the cells and cultured at atemperature of about 30-40EC and a pH of about 5-8 while replacing theculture media with fresh media. From the resulting cultures, the presentpolypeptide can be collected by one or more conventional methods such assalting out, dialysis, filtration, concentration, separatorysedimentation, gel filtration chromatography, ion-exchangechromatography, hydrophobic chromatography, adsorption chromatography,affinity chromatography, chromatofocusing, gel electrophoresis andisoelectrophoresis.

Monoclonal Antibodies and Uses Thereof

As described above, the present polypeptide has a property of inducingINF-γ production by immunocompetent cells, and is expected to be used ina variety of fields as an INF-γ inducer, antiviral agent, antitumoragent, antibacterial agent, immunoregulatory agent, and blood plateletenhancing agent. In general, the development of methods for efficientlypurifying biologically active polypeptides to relatively high purity andfor assaying many samples in parallel are needed when the polypeptidesare to be incorporated into pharmaceuticals. Although the best materialfor enabling purification of and assay for the present polypeptide is amonoclonal antibody, no such monoclonal antibody specific for thepresent polypeptide has been previously established before the presentinvention.

The monoclonal antibody according to the present invention includesmonoclonal antibodies in general which are specific for the presentpolypeptide having the amino acid sequence of SEQ ID NO:6 or of asequence homologous to SEQ ID NO:6, regardless of its source, origin orclass. The homologous amino acid sequence includes those which areobtained by replacing one or more amino acid residues in SEQ ID NO:6with different amino acid residues, by adding one or more amino acidresidues to the N- and/or C-termini or by deleting one or more aminoacid residues from the N- and/or C-termini, while substantially notlosing the activity of inducing the INF-γ production by immunocompetentcells.

The monoclonal antibody according to the present invention can beobtained by using the present polypeptide or its antigenic fragments.For example, the antibody can be obtained by preparing hybridomas usingmammalian cells capable of infinite proliferation and antibody-producingcells collected from mammals immunized with the fragments, selectingclones of hybridomas capable of producing the monoclonal antibody, andculturing the clones in vivo or in vitro.

The present polypeptide as an antigen can be obtained by culturingtransformants into which a DNA encoding the amino acid sequence of SEQID NO:6 or a homologous nucleotide sequence was introduced. Generally,the present polypeptide is used intact or in a partially purified form.The antigenic fragments can be prepared by chemically or enzymaticallyhydrolyzing the wholly purified or partially purified presentpolypeptide, or they can be synthesized by peptide synthesis based onthe amino acid sequence of SEQ ID NO:6.

The immunization method that can be used in the present inventionincludes conventional methods used in the art. For example, antigensalone or in combination with adequate adjuvants are injected intomammals intravenously, intradermally, subcutaneously orintraperitoneally, and they are fed for a prescribed period. Any mammalcan be used in the present invention without special restriction as longas the desired antibody-producing cells can be obtained regardless ofthe animal's species, weight and sex. In general, rodents such as rats,mice and hamsters are used, and the most suitable animal is selectedfrom these rodents while also evaluating compatibility for producinghybridomas with the above mammalian cells capable of infiniteproliferation.

Depending on the species and weight of animal used, the total dose ofantigen is generally in the range of about 5-500 μg per animal and isadministered 2-5 times at an interval of 1-2 weeks. At 3-5 days afterthe final administration, the animal's spleen is extracted and dispersedinto a suspension of spleen cells (antibody-producing cells).

The antibody-producing spleen cells and the mammalian cells capable ofinfinite proliferation are fused to form a cell fusion mixturecontaining the desired hybridomas. Mammalian cells capable of infiniteproliferation include cell strains from mouse myeloma such asP3-NS1-Ag4-1 cells (ATCC TIB18), P3-x63-Ag8 cells (ATCC TIB9),SP2/0-Ag14 cells (ATCC CRL1581), and mutants thereof. Cell fusionmethods that can be used in the present invention include conventionalmethods using an electric pulse and a cell fusion accelerator such aspolyethylene glycol and sendai virus (HVJ). For example,antibody-producing cells and mammalian cells are suspended in fusionmedia containing fusion accelerators in a ratio of about 1:1 to 1:10,and incubated at about 30-40° C. for about 1-5 min. Conventional mediasuch as minimum essential medium (MEM), RPMI 1640 medium, and Iscove'sModified Dulbecco's Medium (IMDM) are preferably used as a fusion mediumwithout the addition of serums such as calf serum.

To select the desired hybridomas, the resultant cell fusion mixture wastransferred to a selection media such as HAT medium, and incubated atabout 30-40° C. for about 3 days to 3 weeks to kill cells that were nothybridomas. Hybridomas were then cultured in the usual manner, and theantibodies secreted by the hybridomas into the culture media wereassayed for reactivity with the present polypeptide. Examples of such anassay are conventional assays for detecting antibodies, such as enzymeimmunoassay, radioimmunoassay, and bioassay.Tan-Clone-Kotai-Jikken-Manual (Experimental Manual for MonoclonalAntibody), edited by Sakuji TOYAMA and Tamie ANDO, published by KodanshaScientific, Ltd., Tokyo, Japan, pp. 105-152 (1991) describes a varietyof suitable assays. Hybridomas which produce antibodies that arespecific to the present polypeptide are readily cloned by using limitingdilution to obtain the hybridoma according to the present invention.

The monoclonal antibody according to the present invention can beobtained by culturing the hybridoma in vivo, i.e., in animals, or invitro. Conventional methods for culturing mammalian cells can be used.In the case of in vivo culture, the monoclonal antibody is collectedfrom the animals' ascites and blood. Examples of such methods includesalting out, dialysis, filtration, concentration, centrifugation,separatory sedimentation gel filtration chromatography, affinitychromatography, high-performance liquid chromatography (HPLC), gelelectrophroesis, and isoelectrophoresis, and, if necessary, two or moreof these techniques can be used in combination. The resultant purifiedmonoclonal antibodies can be concentrated or dried into products in theform of a liquid or a solid depending on their final use.

The present monoclonal antibody is extremely useful for purifying thepresent polypeptide by immunoaffinity chromatography. Such apurification technique includes contacting the monoclonal antibody witha mixture containing the present polypeptide and impurities such asproteins other than the desired polypeptide so as to adsorb the presentpolypeptide on the antibody, and then subsequently desorbing the presentpolypeptide from the antibody after the impurities are removed. Thesesteps are generally carried out in an aqueous system. The monoclonalantibody is generally used in an immobilized form on gel water-insolublecarriers which are packed in cylindrical columns. Cultures oftransformants or their partially purified products are fed to thecolumns to substantially adsorb the present polypeptide to theimmobilized monoclonal antibody. The present polypeptide is then readilydesorbed from the antibody by altering the pH around the antibody. Forexample, in the case of using a monoclonal antibody of the IgG class,the adsorbed polypeptide is desorbed and eluted from the columns at anacidic pH, usually at a pH of 2-3, whereas in the case of a monoclonalantibody of the IgM class, the present polypeptide is desorbed andeluted from the columns at an alkaline pH, usually at a pH of 10-11. Thepurification method according to the present invention attains arelatively high level purification of the present polypeptide with onlythe minimum of labor cost and time. As described above, the presentmonoclonal antibody specifically reacts with the present polypeptidewhich induces INF-γ production by immunocompetent cells. Therefore, themonoclonal antibody is widely used in the purification and detection ofthe present polypeptide and can be prepared in a desired amount by apreparation using hybridomas.

The monoclonal antibody according to the present invention hasrelatively wide applicability to a variety of fields in which thedetection of the present polypeptide is advantageous. When used inlabelled immunoassays such as radioimmunoassay, enzyme immunoassay, andfluorescent immunoassay, the monoclonal antibody can qualitatively andquantitatively detect the present polypeptide in samples instantly andaccurately. In such assays, the monoclonal antibody is labelled, forexample, with radioisotopes, enzymes and/or fluorescent substances priorto use. The antibody specifically reacts with the present polypeptide toexhibit an immunoreaction, and accurately detects a slight amount of thepresent polypeptide in samples by measuring the level of immunoreactionfor these labelled substances. As compared with bioassay, labelledimmunoassay can assay many samples in parallel, reduce the assay timeand labor cost, and provide data with relatively high accuracy. Thus,the present detection method is useful for controlling the productionsteps of the present polypeptide and for quality control of the finalproducts. The techniques for labelling monoclonal antibody or for thelabelling assay are not described in detail because the presentinvention does not in itself relate to such labelling techniques. Thesetechniques are however described in detail in “Enzyme Immunoassay”,edited by P. Tijssen, translated by Eiji ISHIKAWA, published byTokyo-Kagaku-Dojin, pp. 196-348 (1989).

Having now generally described the present invention, the same will bemore readily understood through reference to the following examples,which are provided by way of illustration and is not intended to belimiting of the present invention. The techniques used herein areconventionally known in the art. For example, conventional moleculargenetic techniques are disclosed in Sambrook et al., Molecular Cloning:A Laboratory Manual, second edition, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989, and in Muramatsu, M., LaboratoryManual for Genetic Engineering, Maruzen Co., Ltd., Tokyo, Japan, 1988.

EXAMPLES Example 1 Preparation of Purified Polypeptide

600 female 8 week old CD-1 mice were each injected intraperitoneallywith 1 mg of dead Corynebacterium parvum (ATCC 11827) which had beenpreheated at 60° C. for 1 hour, and the mice were fed in the usualmanner for 7 days and then each was injected intravenously with 1 μg ofa purified lipopolysaccharide derived from Escherichia coli. At 1-2hours after the intravenous injection, the mice were sacrificed bydrawing to collect their blood, followed by removal of their livers,disruption of the livers with a homogenizer in an 8-fold volume of 50 mMphosphate buffer (pH 7.3), and extraction of the resultant suspension.The resultant extract was centrifuged at about 8,000 rpm for 20 min, andapproximately 9 L of the supernatant was admixed with saturated ammoniumsulfate in 50 mM phosphate buffer (pH 7.3) to give a saturation degreeof 45 w/v %. The resultant solution was allowed to stand at 4° C. for 18hours and centrifuged at about 8,000 rpm for 30 min to obtain about 19 Lof supernatant containing the present polypeptide.

The supernatant was fed to a column packed with about 4.6 L of PHENYLSEPHAROSE, a product of Pharmacia LKB Biotechnology AB, Uppsala, Sweden,which had been equilibrated with 50 mM phosphate buffer (pH 7.3)containing 1 M ammonium sulfate. The column was washed with a freshpreparation of the same buffer, and fed at an SV (space velocity) of0.57 with a linear gradient buffer ranging from 1 M to 0.2 M ammoniumsulfate in 50 mM phosphate buffer (pH 7.3). Fractions containing thepresent polypeptide eluted at 0.8 M ammonium sulfate and were collectedand pooled into a volume of about 4.8 L which was then concentrated witha membrane filter, dialyzed against 20 mM phosphate buffer (pH 6.5) at4° C. for 18 hours, and fed to a column packed with about 250 ml ofDEAE-SEPHAROSE, a product of Pharmacia LKB Biotechnology AB, Uppsala,Sweden. The column was washed with a fresh preparation of the samebuffer and fed at an SV of 1.2 with a linear gradient buffer rangingfrom 0 to 0.2 M sodium chloride in 20 mM phosphate buffer (pH 6.5) toelute and collect approximately 260 ml fractions containing the presentpolypeptide eluted at a concentration of about 0.13 M sodium chloride.

Fractions containing the present polypeptide were collected, pooled,concentrated and dialyzed against 25 mM Bis-Tris buffer (pH 7.1) at 4°C. for 18 hours. The dialyzed solution was applied to a column packedwith about 24 ml of MONO-P, a product of Pharmacia LKB Biotechnology AB,Uppsala, Sweden, and eluted with 10 v/v % polybuffer 74 (pH 4.0) whiledecreasing the pH from 7 to 4 to obtain approximately 23 ml of an eluate(pH of about 4.8) containing the present polypeptide. The eluate wasconcentrated, fed to a column packed with SUPERDEX 75, a product ofPharmacia LKB Biotechnology AB, Uppsala, Sweden, which had beenequilibrated with 7 mM phosphate buffer (pH 7.2), 3 mM sodium dihydrogenphosphate, and 139 mM sodium chloride, and subjected to gel filtrationchromatography to elute fractions containing the present polypeptide ofabout 19,000 daltons with a fresh preparation of the same buffer. Thefractions were pooled and concentrated for use in Example 2. The yieldof the present polypeptide was about 0.6 μg/mouse.

Example 2 Partial Amino Acid Sequence

A portion of an aqueous solution containing the purified polypeptide inExample 1 was concentrated to a volume of about 50 μl and then admixedwith 25 μl of a solution containing 3 w/v % SDS, 60 v/v % glycerol, and60 mg/ml dithiothreitol. The resultant mixture was incubated at 50° C.for 30 min, loaded onto 15 w/v % polyacrylamide gel, and electrophoresedin the usual manner. The resultant gel was stained by soaking it in amixture solution of 10 v/v % aqueous acetic acid solution and 50 v/v %aqueous methanol containing 0.1 w/v % Coomassie brilliant blue R 250,destained by repeatedly washing the gel with a mixture solution of 12v/v % aqueous methanol and 7 v/v % aqueous acetic acid solution, andthen washed by soaking it in distilled water for 18 hours. A portion ofthe gel, which was stained with Coomassie brilliant blue and containedthe present polypeptide, was cut out of the gel and lyophilized.

The lyophilized gel was soaked in a 0.6 ml 100 mM sodium hydrogencarbonate solution containing 2 μg/ml TPCK TRYPSIN, 0.5 mM calciumchloride, and 0.02 v/v % aqueous TWEEN 20, followed by incubation at 37°C. for 18 hours to trypsinize the protein. The resultant mixture wascentrifuged to obtain a supernatant, and the resultant precipitate wassoaked in 1 ml of 1 v/v % aqueous trifluoroacetate containing 0.001 v/v% TWEEN 20, shaken for 4 hours at ambient temperature, and centrifugedto obtain a supernatant. The newly formed precipitate was successivelytreated similarly as above with 70 v/v % aqueous trifluoroacetatecontaining 0.001 v/v % TWEEN 20 and with 50 v/v % aqueous acetonitrileto obtain a supernatant. The resultant supernatant and the previouslyobtained supernatant were pooled and concentrated to give 250 μl whichwas then centrifugally filtered.

The resultant peptide fragment-containing aqueous solution was fed to anHPLC ODS-120T column (HPLC column commercialized by Tosoh Corporation,Tokyo, Japan), which had been previously equilibrated with 0.1 v/v %aqueous trifluoroacetate, and the column was washed with 0.1 v/v %aqueous trifluoroacetate, and then fed with 0.1 v/v % trifluoro acetateat a flow rate of 0.5 ml/min while increasing the concentration ofaqueous acetonitrile from 0 to 70 v/v %. The concentration of peptidesin the eluate was monitored by a spectrophotometer at wavelengths of 214nm and 280 nm. Fractions eluted at approximately 75 min and 55 min afterinitiating the elution were collected and respectively designatedpeptide fragment A and peptide fragment B. The elution pattern is shownin FIG. 1.

Peptide fragments A and B were analyzed on a model 473 A proteinsequencer commercialized by Perkin-Elmer Corp., Instrument Division,Norwalk, Conn., and revealed to have the amino acid sequences of SEQ IDNOs:1 and 2.

Example 3 Nucleotide Sequence of CDNA Encoding Polypeptide and AminoAcid Sequence of Encoded Polypeptide

3-1: Preparation of Whole RNA

Three grams of wet mouse liver cells, prepared similarly as in themethod of Example 1, was weighed, soaked in 20 ml of a solutioncontaining 6 M guanidine isothiocyanate, 10 mM sodium citrate, and 0.5w/v SDS, and disrupted with a homogenizer. Thirty-five ml centrifugationtubes, into which 25 ml of 0.1 M EDTA (pH 7.5) containing 5.7 M cesiumchloride was poured, and 10 ml of the homogenized cell suspension wasoverlaid on top of the EDTA-cesium chloride solution in the tubes, wereprepared. The tubes were centrifuged at 25,000 rpm at 20°, for 20 hoursto collect RNA fractions, and the fractions were pooled, distributedinto 15-ml centrifugation tubes, and mixed with equal volumes of amixture of chloroform and 1-butanol (4:1 by volume). The tubes werevibrated/vortexed for 5 min and centrifuged at 4° C. and at 10,000 rpmfor 10 min, and the resulting aqueous phase layers were collected,pooled, and mixed with a 2.5 volumes of ethanol, and allowed to stand at−20° C. for 2 hours to precipitate whole RNA. The precipitate wascollected, pooled, washed with 75 v/v % aqueous ethanol, and dissolvedin 0.5 ml of sterilized distilled water for use in Example 3-2 below.The yield of RNA was about 4 mg, on a dry solid basis (d.s.b.).

3-2: Preparation of cDNA Fragments Encoding Partially the PresentPolypeptide

One μg of whole RNA in Example 3-1 was mixed with 4 μl of 25 mMmagnesium chloride, 2 μl of a solution of 10×PCR buffer consisting of100 mM Tris-HCl buffer (pH 8.3) and 500 mM potassium chloride, 8 μl of 1mM DNTP mix, 1 μl of a solution containing 1 unit/μl RNase inhibitor, 1μl of a solution containing 2.5 units/μl reverse transcriptase, and 1 μlof 2.5 μM random hexamer, and further mixed with water to give a totalvolume of 20 μl. The mixed solution was placed in 0.5 ml reaction tubes,and, in the usual manner, successively incubated at 25° C. for 10 min,at 42° C. for 30 min, at 99° C. for 5 min, and at 5° C. for 5 min toeffect the reverse transcriptase reaction, followed by recovering anaqueous solution containing the first strand cDNA.

To 20 μl of the aqueous solution, 4 μl of 25 mM magnesium chloride, 8 μlof 10×PCR buffer, 0.5 μl of a solution containing 2.5 units/μl ofAmpliTaq DNA polymerase commercialized by Perkin-Elmer Corp., InstrumentDiv., Norwalk, Conn., USA, and 1 pmol each of primers 1 and 2 as a senseprimer or an anti-sense primer were added. The mixture was made up to avolume of 100 μl with sterilized distilled water, and, in the usualmanner, successively incubated at 94° C. for 1 min, at 45° C. for 2 min,at 72° C. for 3 min in a cyclic manner for 40 cycles to amplify a DNAfragment, which partially encodes the present polypeptide, by using thefirst strand cDNA as a template. Primers 1 and 2 were oligonucleotideswhich were chemically synthesized based on the amino acid sequences ofPro-Glu-Asn-Ile-Asp-Asp-Ile (corresponding to residues 10-16 of SEQ IDNO:1) and Phe-Glu-Asp-Met-Thr-Asp-Ile (corresponding to residues 4-10 ofSEQ ID NO:2) and have the nucleotide sequences of5′-ATRTCRTCDATRTTYTCNGG-3′ (SEQ ID NO:9) and 5′-TTYGARGAYATGACNGAYAT-3′(SEQ ID NO:10).

A portion of the resultant PCR product was fractionated byelectrophoresis on a 2 w/v % agarose gel, transferred onto a nylonmembrane, fixed with 0.4 N sodium hydroxide, washed with 2×SSC, airdried, soaked in a prehybridization solution containing 5×SSPE, 5×Denhardt's solution, 0.5 w/v % SDS and 100 μg/ml of denatured salmonsperm DNA, and incubated at 65° C. for 3 hours. An oligonucleotide probe1 having a nucleotide sequence of 5′TTYGARGARATGGAYCC-3′ (SEQ ID NO:11)was synthesized based on the amino acid sequence ofPhe-Glu-Glu-Met-Asp-Pro in (corresponding to residues 4-9 of SEQ IDNO:1), and labeled with [γ-³²P]ATP and T4 polynucleotide kinase. Thenylon membrane was soaked in a solution containing 1 pmole of probe 1,5×SSPE, 5 × Denhardt's solution, 0.5 w/v % SDS, and 100 μg/ml of adenatured salmon sperm DNA, and incubated at 45° C. for 24 hours toeffect hybridization. The resultant nylon membrane was washed with 6×SSCand autoradiographed in the usual manner, revealing that the PCR productcontained the desired DNA fragment.

The remaining PCR product was mixed with 50 ng of the pT7 BLUE T plasmidvector commercialized by Takara Shuzo Co., Ltd., Tokyo, Japan, anadequate amount of T4 ligase, and further mixed with 100 mM ATP to givea concentration of 1 mM, followed by incubation at 16° C. for 18 hoursto insert the DNA fragment into the plasmid vector. The recombinant DNAthus obtained was introduced by the competent cell method intoEscherichia coli NoVa Blue strain, a microorganism of the speciesEscherichia coli commercialized by Pharmacia LKB Biotechnology AB,Uppsala, Sweden, in order to obtain a transformant which was theninoculated onto an agar plate containing 10 g/l bactotryptone, 2.5 g/lsodium chloride, 15 g/l bacto-agar, 100 mg/l ampicillin, 40 mg/l X-Galand 23.8 mg/l isopropyl-β-D-thiogalacto-pyranoside (abbreviatedhereinafter as IPTG), and incubated at 37° C. for 24 hours to formcolonies. A nylon membrane was overlaid on an agar medium plate in theusual manner and allowed to stand for about 30 seconds to attach thecolonies to the nylon membrane. The nylon membrane was then detachedfrom the plate and soaked for 7 min in a solution containing 0.5 Nsodium hydroxide and 1.5 M sodium chloride to effect cell lysis.Thereafter, the nylon membrane was further soaked for 3 min in 0.5 MTris-HCl buffer (pH 7.2) containing 1.5 M sodium chloride, washed with2×SSC, soaked in 0.4 N sodium hydroxide for 20 min to fix the DNA,washed with 5×SSC, air-dried, soaked in a prehybridization solutioncontaining 5×SSPE, 5 × Denhardt's solution, 0.5 w/v % SDS, and 100 μg/mldenatured salmon sperm DNA, and incubated at 65° C. for 3 hours. Thecolonies formed on the nylon membrane were hybridized with probe 1,washed with 6×SSC, and autoradiographed similarly as above, followed byselecting transformants which strongly hybridized to probe 1.

The transformants were inoculated into L-broth (pH 7.2) containing 100μg/ml ampicillin and incubated at 37° C. for 18 hours, followed bycollecting cells from the culture and isolating recombinant DNA by theconventional alkaline-SDS method. Analysis by the dideoxy method ofnucleotide sequencing revealed that the recombinant DNA contained a DNAfragment consisting of the nucleotide sequence corresponding tonucleotides 85 to 281 of SEQ ID NO:3.

3-3: Preparation of mRNA

0.05 ml of an aqueous solution containing whole RNA in Example 3-1 wasplaced in a test tube, admixed with 0.5 ml of 10 mM Tris-HCl buffer (pH7.5) containing 1 mM EDTA and 0.1 w/v % SDS, and made up to a volume of1 ml with sterilized distilled water. To the mixture was added 1 mlOLIGOTEX-dT30 SUPER, an oligo-(dT)₃₀ latex commercialized by NipponRoche K.K., Tokyo, Japan, followed by incubation at 65° C. for 5 min todenature the RNA and then cooling for 3 min in an ice-chilled bath. Theresultant mixture was admixed with 0.2 ml of 5 M sodium chloride,incubated at 37° C. for 10 min, and centrifuged at 10,000 rpm at 25° C.for 10 min. The precipitate in the form of a pellet was suspended in 0.5ml sterilized distilled water, and incubated at 65° C. for 5 min toextract mRNA from the oligo-(dT)₃₀ latex. The yield of the mRNA wasabout 5 μg.

3-4: Preparation of cDNA Library

A cDNA Library was prepared from the mRNA in Example 3-3 by using cDNASYNTHESIZING SYSTEM PLUS, a cDNA cloning kit commercialized by AmershamCorp., Amersham International, Arlington Heights, Ill., USA. Theprocedures used are as follows: To a 1.5-ml reaction tube weresuccessively added 4 μl of a solution for synthesizing the first strandcDNA, 1 μl sodium pyrophosphate solution, 1 μl of a solution of humanplacenta RNase inhibitor, 2 μl dNTP mix, and 1 μl oligo-(dT)₁₆ primer.The resultant mixture was mixed with 2 μg of mRNA from Example 3-3, madeup to a volume of 19 μl with sterilized distilled water, mixed with 1 μlof a solution containing 20 units of reverse transcriptase, andincubated at 42° C. for 40 min to obtain a reaction mixture containingthe first strand cDNA.

The reaction mixture obtained was mixed with 37.5 μl of a solution forsynthesizing the second strand cDNA, 0.8 unit of ribonuclease H derivedfrom Escherichia coli, 23 units of DNA polymerase I, and made up to avolume of 100 μl with sterilized distilled water. The resultant mixturewas successively incubated at 12° C. for 60 min and at 22° C. for 60min, mixed with 2 units of T4 DNA polymerase, and incubated at 37° C.for 10 min to obtain a reaction mixture containing the second strandcDNA. To the reaction mixture was added 4 μl of 0.25 M EDTA (pH 8.0) toterminate the reaction, and the resultant mixture was extracted withphenol/chloroform and treated with ethanol in the usual manner toprecipitate the cDNA, followed by recovering the cDNA in theprecipitate.

To the CDNA thus obtained were added in the following order: 2 μl of L/Kbuffer, 250 pmol EcoRI adaptor, and 2.5 units of T4 DNA ligase in thisorder. The resultant solution was made up to a volume of 20 μl withsterilized distilled water, and incubated at 15° C. for 16 hours toligate the EcoRI adaptor to both ends of the CDNA. The reaction mixturewas mixed with 2 μl of 0.25 M EDTA to inactivate the remaining enzyme,and subjected to molecular sieve chromatography to remove intact EcoRIadaptor. 40 μl of L/K buffer and 80 units of T4 polynucleotide kinasewere added to the mixture, and the mixture was made up to a volume of400 μl with sterilized distilled water, followed by incubation at 37° C.for 30 min to methylate the EcoRI cleavage sites. The resultant mixturewas extracted with phenol/chloroform and treated with ethanol toprecipitate the objective DNA, followed by recovery of the DNA. To theDNA were added 1.5 μl of L/K buffer containing an adequate amount ofλgt10 arms, and 2.5 units of T4 DNA ligase. The resultant solution wasmade up to a volume of 15 μl with sterilized distilled water, incubatedat 15° C. for 16 hours to effect ligation, and subjected to conventionalin vitro packaging method to obtain a phage containing a recombinantλDNA.

3-5: Cloning of Recombinant DNA

A seed culture of Escherichia coli NM514 strain was infected with thephage from Example 3-4 in the usual manner, and the infected cells wereinoculated onto an agar plate (pH 7.0) containing 10 g/l bacto-tryptone,5 g/l bacto-yeast extract, 10 g/l sodium chloride and 15 g/l bacto-agar,and incubated at 37° C. for 16 hours to form plaques. The agar plate wascovered with a nylon membrane and allowed to stand for about 30 secondsto attach the plaques to the nylon membrane. The nylon membrane was thendetached from the plate, and successively soaked in an aqueous solutioncontaining 0.5 M sodium hydroxide and 1.5 M sodium chloride for 7 minand in 0.5 M Tris-HCl buffer (pH 7.0) containing 1.5 M sodium chloridefor 3 min. The nylon membrane was washed with 2×SSC, air dried, soakedin a solution containing 5×SSPE, 5× Denhardt's solution, 0.5 w/v % SDS,and 100 μg/ml denatured salmon sperm DNA, and incubated at 65° C. for 3hours. Thereafter, the resultant nylon membrane was incubated in asolution containing an adequate amount of the DNA fragment obtained inExample 3-2 as probe 2 and labeled with ³²P by the READY PRIME DNALABELLING SYSTEM, a DNA labeling kit commercialized by Amersham Corp.,Amersham International, Arlington Heights, Ill., USA, in 5×SSPE, 5×Denhardt's solution, 0.5 w/v % SDS, and 100 μg/ml of denatured salmonsperm DNA, and incubated at 60° C. for 20 hours to effect hybridization.The resultant hybridized nylon membrane was subjected toautoradiography, similar to what was done above, to select phage DNAclones which strongly hybridized with probe 2.

Using conventional techniques, the clones were amplified in Escherichiacoli, followed by extracting recombinant DNA from the cells. Therecombinant DNA was then digested with the restriction enzyme EcoRI.Plasmid vector pUCl9 (ATCC 37254) was also digested with the samerestriction enzyme, and the resultant digested DNA fragments and plasmidfragments were ligated together with DNA ligase to obtain a recombinantDNA which was then introduced into Escherichia coli JM109 strain (ATCC53323) by the conventional competent cell method to obtain a transformedhost cell.

3-6: Determination of Nucleotide and Amino Acid Sequences

The transformed E. coli host cell obtained in Example 3-5 was inoculatedinto L-broth (pH 7.2) and cultured at 37° C. for 13 hours withagitation. The resultant proliferated cells were collected and treatedaccording to the conventional alkaline-SDS method to obtain arecombinant DNA containing the DNA according to the present invention.Analysis on an automatic sequencer using a fluorophotometer revealedthat the recombinant DNA contains the nucleotide sequence from the5′-terminus in SEQ ID NO:3. Further analysis of the nucleotide sequenceindicated that it encodes the amino acid sequence containing theN-terminus in SEQ ID NO:4. The amino acid sequence contains the partialamino acid sequences of SEQ ID NOs:1 and 2 corresponding to residues 79to 103 and from 26 to 43 in SEQ ID NO:4, and this means that the presentpolypeptide includes the amino acid sequence containing the N-terminusof SEQ ID NO:4, and that it is encoded by a DNA containing thenucleotide sequence from the 5′-terminus of SEQ ID NO:3.

In the following Examples 4 to 7, a cDNA, which encodes anotherpolypeptide that induces the IFN-γ production by immunocompetent cells,is prepared from human liver mRNA by using a DNA fragment of thenucleotide sequence of SEQ ID NO:3 as probe. The nucleotide sequence ofthe cDNA was analyzed and was determined to encode the amino acidsequence of the polypeptide. The cDNA was expressed and produced inEscherichia coli, and the features and properties of the polypeptideproduced were studied.

Example 4 Nucleotide and Amino Acid Sequence of Polypeptide

4-1: Preparation of cDNA Library

A cDNA library was prepared from normal human liver tissue poly(A)⁺toRNA, a product commercialized by ClonatecBIOSOFT, Paris Cedex, France,by using a cDNA cloning kit, cDNA SYNTHESIZING SYSTEM PLUS,commercialized by Amersham Corp., Amersham International, ArlingtonHeights, Ill., USA. The procedures used are as follows: To a 1.5 mlreaction tube were successively added 10 μl of a solution forsynthesizing the first strand cDNA, 2.5 μl of 1 mM sodium pyrophosphate,2.5 μl of a solution containing 1 μg/μl of a human placenta ribonucleaseinhibitor, 5 μl of a solution containing 1 μg/μl of a dNTP mix, 2.5 μlof a solution containing 1 μg/μl oligo-dT primer, 5 μg of normal humanliver tissue poly(A)⁺ RNA, and made up to a volume of 45 μl withsterilized distilled water. Thereafter, the resultant mixture was mixedwith 5 μl of a solution containing reverse transcriptase, and incubatedat 42° C. for 40 min to obtain a reaction mixture containing the firststrand cDNA.

To the reaction mixture was added 93.5 μl of a solution for synthesizingthe second strand cDNA, 4 units of RNaseH derived from Escherichia coli,115 units of DNA polymerase, and made up to a volume of 250 μl withsterilized distilled water. The resultant mixture was successivelyincubated at 12° C. for 60 min, at 22° C. for 60 min, and at 70° C. for10 min, mixed with 10 units of T4 polymerase, and further incubated at37° C. for 10 min. To the reaction mixture was added 10 μl of 0.25 MEDTA (pH 8.0) to terminate the reaction, and the resultant mixture wasextracted with phenol/chloroform, and treated with ethanol toprecipitate the objective double stranded cDNA in the usual manner,followed by recovering the precipitate.

To the double stranded cDNA thus obtained were added 2 μl L/K buffer (pH8.0), 250 pmol EcoRI adaptor, and 2.5 units of T4 DNA ligase, and theresultant solution was made up to a volume of 20 μl with sterilizeddistilled water, and incubated at 15° C. for 16 hours to ligate theEcoRI adaptor to both ends of the cDNA. The resultant mixture was thenmixed with 2 μl of 0.25 M EDTA to terminate the reaction, and subjectedto molecular sieve chromatography to remove unligated EcoRI adaptor. 40μl of L/K buffer (pH 8.0) and 80 units of T4 polynucleotide kinase wereadded to the resultant mixture and the mixture was made up to a volumeof 400 μl with sterilized distilled water, followed by incubation at 37°C. for 30 min to methylate the EcoRI cleavage sites. The resultantmixture was extracted with phenol/chloroform and treated with ethanol toprecipitate the objective cDNA, followed by recovering the cDNA. To thecDNA were added 1.5 μl of L/K buffer (pH 8.0) containing an adequateamount of λgt10 arms, and 2.5 units of T4 DNA ligase, and the resultantsolution was made up to a volume of 15 μl with sterilized distilledwater, incubated at 15° C. for 16 hours to effect ligation, andsubjected to conventional in vitro packaging method to obtain a phagecontaining recombinant λDNA.

4-2: Cloning of Recombinant DNA

A seed culture of Escherichia coli NM514 strain was infected with thephage in Example 4-1, and the infected cells were inoculated onto anagar plate (pH 7.0) containing 10 g/l bacto-tryptone, 5 g/l bacto-yeastextract, 10 g/l sodium chloride, and 15 g/l bacto-agar, and incubated at37° C. for 16 hours to form plaques. According to conventional methods,the agar plate was covered with a nylon membrane and allowed to standfor about 30 seconds to attach the plaques thereto. Thereafter, thenylon membrane was detached from the plate, and successively soaked inan aqueous solution containing 0.5 N sodium hydroxide and 1.5 M sodiumchloride for 7 min and in 0.5 M Tris-HCl buffer (pH 7.0) containing 1.5M sodium chloride for 3 min. The nylon membrane was washed with 5×SSC,air-dried, soaked in a solution containing 5×SSPE, 5× Denhardt'ssolution, 0.5 w/v % SDS and denatured salmon sperm DNA, and incubated at65° C. for 3 hours. To clone the objective recombinant DNA, a DNAfragment having the nucleotide sequence of SEQ ID NO:3 was labeled with³²P using the READY PRIME DNA LABELLING SYSTEM, a DNA labeling kitcommercialized by Amersham Corp., Amersham International, ArlingtonHeights, USA, and designated as probe 3. The procedures used are asfollows: Place 25 ng of a DNA fragment prepared by the method in Example3-5 in a 1.5 ml reaction tube, made up to a volume of 45 μl withsterilized distilled water, incubated at 95° C. for 3 min, andtransferred to another reaction tube. Five μl of an [α-³²P]dCTP solutionwas added to the reaction tube, and the DNA fragment was labeledaccording to the instruction attached to the kit by incubating it at 37°C. for 30 min. Thereafter, the resultant product containing the labeledDNA fragment was subjected to conventional molecular sievechromatography to remove unincorporated [α-³²P] dCTP.

The above nylon membrane was soaked in a solution containing 5×SSPE, 5×Denhardt's solution, 0.5 w/v % SDS, and 100 μg/ml of a denatured salmonsperm DNA, and the mixture was incubated at 60° C. for 20 hours toeffect hybridization, and further washed at ambient temperature in 6×SSCfor 20 min and in 2×SSC for 20 min. The resultant hybridized nylonmembrane was subjected to autoradiography to select phage DNA cloneswhich strongly hybridized with probe 3.

With conventional techniques, the DNA clones were amplified inEscherichia coli, and the recombinant DNA was extracted from the cells.The recombinant DNA was digested with the restriction enzyme EcoRI.Plasmid vector pUCl9 (ATCC 37254) was digested with the same restrictionenzyme, and the cleaved DNA fragments and plasmid fragments were ligatedwith DNA ligase to obtain a recombinant DNA which was then introducedinto Escherichia coli JM109 strain (ATCC 53323) by the conventionalcompetent cell method to obtain a transformant containing the presentDNA.

4-3: Determination of Nucleotide Sequence and Amino Acid Sequence

The transformant obtained in Example 4-2 was inoculated into L-broth (pH7.2) containing 50 μg/ml of ampicillin, and cultured at 37° C. for 18hours with agitation. The proliferated cells were collected bycentrifugation and treated according to the conventional alkaline-SDSmethod to extract the recombinant DNA. The analysis of the nucleotidesequence on an automatic sequencer using a fluorophotometer revealedthat the recombinant DNA contains the nucleotide sequence of SEQ IDNO:7. The amino acid sequence deduced from the nucleotide sequence isshown as SEQ ID NO:8, indicating that the present polypeptide includesthe amino acid sequence of SEQ ID NO:6, and that the polypeptide isencoded by the DNA of nucleotide sequence SEQ ID NO:5. In SEQ ID NO:8,Xaa represents isoleucine or threonine.

Example 5 Preparation of Replicable Recombinant DNA and Transformants

To a 0.5 ml reaction tube were added 8 μl of 25 mM magnesium chloride,10 μl of 10×PCR buffer, 8 μl of 1 mM dNTP mix, 0.5 μl of a solutioncontaining 2.5 units/μl AmpliTaq DNA polymerase, and 1 ng of therecombinant DNA of Example 4-2. The resultant mixture was mixed withadequate amounts of two oligonucleotides, as a sense primer or ananti-sense primer, having the nucleotide sequences represented by5′-CGAGGGATCCTACTTTGGCAAGCTTG-3′ (SEQ ID NO:12) and5′-CAAGGAATTCCTAGTCTTCGTTTTG-3′ (SEQ ID NO:13), which had beenchemically synthesized based on the nucleotide sequences encoding theamino acid residues near the N- and C-termini in SEQ ID NO:5, and madeup to a volume of 100 μl with sterilized distilled water. The resultantmixture was successively incubated at 94° C. for 1 min, at 60° C. for 2min, and at 72° C. for 3 min, and this incubation cycle was repeated for40 times to obtain a PCR product which was then digested withrestriction enzymes BamHI and EcoRI to obtain a BamHI-EcoRI DNAfragment. The resultant BamHI-EcoRI DNA fragment was mixed with anadequate amount of sterilized distilled water. The solution was thenmixed with 10 ng of a plasmid vector, pGEX-2T (commercialized byPharmacia LKB Biotechnology AB, Uppsala, Sweden), which had beenpreviously digested with BamHI and EcoRI as restriction enzymes, 10 μlof 10× ligation buffer, and an adequate amount of T4 DNA ligase and 10mM ATP to give a final concentration of 1 mM, followed by incubation at16° C. for 18 hours to obtain the replicable recombinant DNA, pHIGIF.

The recombinant DNA PHIGIF was introduced by conventional competent cellmethod into Escherichia coli DH5α strain commercialized by Toyobo Co.,Ltd., Tokyo, Japan, and the resultant transformant HIGIF was inoculatedinto L-broth (pH 7.2) containing 50 μg/ml ampicillin, and incubated at37° C. for 18 hours with agitation. The resultant culture wascentrifuged to obtain the proliferated transformants, which were thensubjected to conventional alkaline-SDS method to extract the recombinantDNA pHIGIF. The analysis of the recombinant PHIGIF by the dideoxy methodrevealed that as shown in FIG. 2, HIGIF cDNA or the cDNA in SEQ ID NO:5are ligated into the sites downstream of the sequences for the Tacpromotor and glutathione S-transferase gene.

Example 6 Production of Polypeptide from the Transformant

The transformant HIGIF from Example 5 was inoculated into T-broth (pH7.2) containing 50 μg/ml of ampicillin, and incubated at 37° C. for 18hours with agitation to obtain a seed culture. Eighteen L aliquots of afresh preparation of T-broth (pH 7.2) were placed in 30-L jarfermenters, inoculated with 1 v/v % of the seed culture, and cultured at37° C. under aeration agitation conditions. During cultivation, theculture was sampled and monitored for absorbance at a wavelength of 650nm, and when the absorbance reached approximately 1.5, IPTG was added tothe culture to give a concentration of 0.1 mM. Thereafter, the culturewas further incubated for another 5 hours and centrifuged to separatecells from the culture. The cells were suspended in a solution (pH 7.2)containing 139 mM sodium chloride, 7 mM disodium hydrogen phosphate, and3 mM sodium dihydrogen phosphate, treated with ultrasound in the usualmanner and centrifuged to obtain a supernatant.

The supernatant was fed to a column packed with GLUTATHIONE SEPHAROSE4B, a product of Pharmacia LKB Biotechnology AB, Uppsala, Sweden, whichhad been previously equilibrated with a solution (pH 7.2) containing 139mM sodium chloride, 7 mM disodium hydrogen phosphate and 3 mM sodiumdihydrogen phosphate. The column was washed with a fresh preparation ofthe same solution, and 100 U of thrombin was added to 1 ml of the gel inthe column to effect an enzymatic cleavage reaction while allowing thecolumn to stand at ambient temperature for 16 hours. The column was fedwith a fresh preparation of the same solution above to elute thereaction product, and the eluate was fed to a column packed withSUPERDEX 75, a product of Pharmacia LKB Biotechnology AB, Uppsala,Sweden, followed by collecting fractions corresponding in size to about18,500 daltons. The fractions were pooled, concentrated and lyophilizedto obtain a solid product containing the present polypeptide in a yieldof about 80 μg per L of culture.

Example 7 Physicochemical Property of Polypeptide

7-1: Molecular Weight

In accordance with the method reported by U. K. Laemmli in Nature,227:680-685 (1970), the purified polypeptide prepared in Example 6 waselectrophoresed on a sodium dodecyl sulfate (SDS) polyacrylamide gelfree of reducing agent to show mainly a single protein band with IFN-γinducibility at a position corresponding to about 18,500±3,000 daltons.The marker proteins used in this experiment were calf serum albumin(MW=67,000 daltons), ovalbumin (MW=45,000 daltons), soy bean trypsininhibitor (MW=20,100 daltons), and α-lactalbumin (MW=14,400 daltons).

7-2: Isoelectric Point

The purified polypeptide in Example 6 was chromatofocused and exhibitedan isoelectric point of about 4.9±1.0.

7-3: Amino Acid Sequence Containing the N-terminus

The purified polypeptide from Example 6 was analyzed on a MODEL 473 Aprotein sequencer commercialized by Perkin-Elmer Corp., Instrument Div.,Norwalk, Conn., and revealed that it had the structure where a peptide,“Gly-Ser-”, is coupled to the tyrosine residue at the N-terminus of theamino acid sequence of SEQ ID NO:14 by the fusion with glutathioneS-transferase and by the cleavage with thrombin.

7-4(a): Biological Activity

Spleen cells from 8 week old female C3H/HeJ mice were excised andsuspended in serum-free RPMI 1640 medium (pH 7.4), and the resultantcells were washed with a fresh preparation of the same medium, andsoaked in Gey solution (pH 8.0) to effect hemolysis. The resultantspleen cells were suspended in RPMI 1640 medium (pH 7.4) supplementedwith 10 v/v % fetal serum to give a cell density of 1×10⁷ cells/ml. Tenml aliquots of the cell suspension were distributed into plastic petridishes, 9 cm in diameter, and incubated at 37° C. for 1 hour in a 5 v/v% CO₂ incubator. Only cells floating in the resultant cultures werecollected and washed with RPMI 1640 medium (pH 7.4) supplemented with 10v/v % fetal bovine serum for use in the following test for IFN-γinduction.

Mouse spleen cells were suspended in RPMI 1640 medium (pH 7.4)supplemented with 10 v/v % fetal calf serum to give a cell density of1×10⁷ cells/ml, and 0.15 ml aliquots of which were distributed to96-well microplates. 0.05 ml of a solution of a purified polypeptidefrom Example 6 diluted with a fresh preparation of the same medium wasadded to each well, and the cells were incubated with or without theaddition of 0.05 ml of 2.5 μg/ml concanavalin A or 50 units/mlinterleukin 2, at 37° C. for 24 hours in a 5 v/v % CO₂ incubator. Aftercompletion of the culture, 0.1 ml of the resultant supernatant in eachwell was sampled to assay the activity of the formed IFN-γ by enzymeimmunoassay. As a control, a system similar to the system above wasprovided and similarly treated as above except that the purifiedpolypeptide, concanavalin A and interleukin 2 were not used. As an IFN-γstandard, a mouse IFN-γ preparation Gg02-901-533, obtained from theNational Institutes of Health, Bethesda, Md., USA was used and theactivity of IFN-γ was expressed in international units (IU). The resultsare shown in Table 1.

TABLE 1 IFN-γ production by mouse spleen cell (IU/ml) Sampleconcentration Sample plus Sample plus (μg/ml) Sample concanavalin Ainterleukin 2 10.00 12 138 118 3.33 6 88 55 1.11 5 56 16 0.37 5 21 120.12 5 12 10 0.04 5 11 7 0 0 4 1 Note: In the Table, Sample means thepresent polypeptide.7-4(b): Induction of IFN-γ Production from Human Lymphocyte

By using a syringe containing heparin, blood was collected from ahealthy donor, which was then diluted two-fold with serum-free RPMI 1640medium (pH 7.4), and overlaid onto FICOLL in a centrifuge tube.Lymphocytes obtained by centrifugation at 2,000 rpm for 20 min were thenwashed with RPMI 1640 medium (pH 7.4) supplemented with 10 v/v % fetalcalf serum, suspended in a fresh preparation of the same medium to givea cell density of 5×10⁶ cells/ml, and treated similarly as in Example7-4(a) except that a human IFN-γ standard, Gg23-901-530, obtained fromthe National Institutes of Health, Bethesda, Md., USA, was used as anIFN-γ standard. The results are shown in Table 2.

TABLE 2 IFN-γ production by human lymphocyte (IU/ml) Sampleconcentration Sample plus Sample plus (μg/ml) Sample concanavalin Ainterleukin 2 10.00 191 479 1,182 3.33 169 576 1,419 1.11 168 426 1,1060.37 150 296 739 0.12 74 193 390 0.04 36 137 324 0 1 11 24 Note: In theTable, Sample means the present polypeptide.

The results in Tables 1 and 2 demonstrate that the present polypeptidehas an activity of inducing IFN-γ production by immunocompetent cells ofmammals including human and mouse. In the control groups, no significantIFN-γ production was found, while in the systems in which the presentpolypeptide was present, significant IFN-γ production was observed. Thisactivity of the polypeptide was strongly augmented when used incombination with concanavalin A or interleukin 2 as a cofactor.

Example 8 Preparation of Polypeptide

The immunoreaction of newborn hamsters were suppressed in a conventionalmanner by injecting a rabbit antiserum against hamster thymus into thehamsters, transplanting to their dorsal subcutaneous tissues with about5×10⁵ cells/hamster of THP-1 cells (ATCC TIB202), a myelomonocytic cellline of a human acute monocytic leukemia, and fed for three weeks in aconventional manner. Tumor masses that formed in subcutaneous tissues,about 15 g by weight per hamster, were extracted, dispersed in aconventional manner in physiological saline, and washed with phosphatebuffered saline (PBS).

The propagated cells thus obtained were washed with ten volumes of cold20 mM HEPES buffer (pH 7.4) containing 10 mM potassium chloride, 1.5 mMmagnesium chloride, and 0.1 mM EDTA, allowed to stand in three volumesof a fresh preparation of the same buffer under ice chilled conditions,frozen at −80° C., and thawed to disrupt the cells. The disrupted cellswere centrifuged to obtain a supernatant which was then fed to a columnpacked with DEAE-SEPHAROSE (a gel for ion-exchange column chromatographycommercialized by Pharmacia LKB Biotechnology AB, Uppsala, Sweden)previously equilibrated with 10 mM phosphate buffer (pH 6.6), followedby washing the column with 10 mM phosphate buffer (pH 6.6), eluting witha sodium chloride which is increased stepwise from 0 M to 0.5 M in 10 mMphosphate buffer (pH 6.6), and collecting a fraction which elutes atabout 0.2 M sodium chloride.

The collected fraction was dialyzed against 10 mM phosphate buffer (pH6.8) and fed to a column packed with DEAE 5PW previously equilibratedwith the buffer (a gel for ion-exchange chromatography commercialized byTosoh Corporation, Tokyo, Japan), followed by eluting with a sodiumchloride gradient which was increased from 0 M to 0.5 M in 10 mMphosphate buffer (pH 6.8), and collecting fractions which eluted atabout 0.2-0.3 M sodium chloride.

The collected fractions were pooled, dialyzed against PBS, fed to aplastic cylindrical immunoaffinity chromatography column packed with agel containing a monoclonal antibody which had been prepared accordingto the method disclosed in Japanese Patent Application No.58,240/95(applied for by the present applicant and which corresponds to U.S. Ser.No. 08/555,818), and washed with PBS. The column was fed with 100 mMglycine-HCl buffer (pH 2.5) to collect eluate fractions which containeda polypeptide that induces IFN-γ production by immunocompetent cells.The fractions collected were pooled, dialyzed against sterile distilledwater, concentrated with a membrane filter, and lyophilized to obtain apurified solid polypeptide with a yield of about 50 ng per hamster.

Example 9 Molecular Weight

In accordance with the method reported by U. K. Laemmli, Nature,227:680-685 (1970), a purified polypeptide prepared by the methoddisclosed in Example 8 was electrophoresed on SDS-PAGE in the presenceof 2 w/v % dithiothreitol, and a main protein band with an IFN-γinducibility was observed at a position corresponding to a molecularweight of about 18,000-19,500 daltons. The proteins used as markers werebovine serum albumin (MW=67,000 daltons), ovalbumin (MW=45,000 daltons),carbonic anhydrase (MW=30,000 daltons), soy bean trypsin inhibitor(MW=20,100 daltons), and α-lactalbumin (MW=14,400 daltons).

Example 10 Amino Acid Sequence and Peptide Mapping in the N-terminalRegion

10-1: Amino Acid Sequence Near at the N-terminus

The purified polypeptide of Example 8 was analyzed on a MODEL 473Aprotein sequencer commercialized by Perkin-Elmer Corp., Norwalk, Conn.,and it was found to have the amino acid sequence of SEQ ID NO:14, and inparticular, SEQ ID NO:18.

10-2: Peptide Mapping

A purified polypeptide obtained by the method in Example 8 was dissolvedin an adequate amount of sterile distilled water, and the solution wasfed to a column packed with ASAHIPAK® C4P-50 4E (a gel forhigh-performance liquid chromatography (HPLC) commercialized by ShowaDenko, K.K., Tokyo, Japan) which had been previously equilibrated with0.1 v/v % aqueous trifluoroacetic acid solution. The column was washedwith 0.1 v/v % aqueous trifluoroacetic acid solution and a lineargradient solution of acetonitrile from 0 v/v % to 90 v/v % in a solutionof a mixture of trifluoroacetic acid and acetonitrile was fed to thecolumn at a flow rate of 60 ml/hour. Eluted fractions containing apolypeptide which induces the IFN-γ production by immunocompetent cellswere collected, pooled, neutralized with 1 M aqueous Tris solution (pH11.2), and concentrated in a conventional manner. To 50 mM Tris-HClbuffer (pH 8.5), in which was dissolved an adequate amount ofclostripain (commercialized by Sigma Chemical Company, St. Louis, Mo.,USA), the polypeptide was added in an amount of about 50 times that ofclostripain on the basis of molar ratio while removing acetonitrile, andthe resulting mixture was allowed to react at a pH of 8-9 and at 37° C.for 12 hours to obtain a reaction mixture containing fragments of thepolypeptide.

The reaction mixture was fed to a column packed with ODS-120T (a gel forHPLC commercialized by Tosoh Corporation, Tokyo, Japan), which had beenpreviously equilibrated with 0.1 v/v % aqueous trifluoroacetic acidsolution. The column was washed with 0.1 v/v % aqueous trifluoroaceticacid solution and a linear gradient solution of acetonitrile from 0 v/v% to 70 v/v % in a solution of a mixture of trifluoroacetic acid,acetonitrile and water (where the concentration of trifluoroacetic acidwas 0.09 v/v %) was fed at a flow rate of 30 ml/hour while monitoringthe absorption level of the peptide, i.e., the concentration of thepeptide, at a wave length of 214 nm. FIG. 3 shows the resulting peptidemap.

In FIG. 3, peptide fragments eluted at about 59, 62 and 68 min afterinitiating the elution are designated peptide fragments 1, 2 and 3,respectively. These peptide fragments were separately collected andanalyzed for amino acid sequence on MODEL 473A, a protein sequencercommercialized by Perkin-Elmer Corp., Norwalk, Conn., in a conventionalmanner. As a result, it was found that peptide fragments 1 and 2 havethe amino acid sequences of SEQ ID NOs:15 and 19, respectively, andpeptide fragment 3 has the amino acid sequences of SEQ ID NOs:16 and 17.Comparison of these amino acid sequences with SEQ ID NO:6 revealed thatpeptide fragments 1 to 3 correspond to the residue positions 148-157,1-13, and 45-58 and 80-96, respectively, in the amino acid sequence ofSEQ ID NO:6. These results confirmed that polypeptide fragments 1 and 2correspond to the C- and N-terminal fragments of the polypeptide usedfor analysis, and that polypeptide fragment 3 corresponds to an internalfragment of the polypeptide.

It is concluded that the purified polypeptide obtained by the method inExample 8 contains the amino acid sequence of SEQ ID NO:6 becauseExample 9 found that the purified polypeptide has a main protein band ata position corresponding to a molecular weight of about 18,000-19,500daltons on SDS-PAGE, and the purified polypeptide is calculated to havea molecular weight of 18,199 daltons from the amino acid sequence of SEQID NO:6.

Example 11 Biological Activity

11-1: IFN-γ Production by Immunocompetent Cell

A blood sample taken from a healthy volunteer with a heparinized syringewas diluted two-fold with serum-free RPMI 1640 medium (pH 7.4). Thediluted blood was overlaid onto FICOLL (commercialized by Pharmacia LKBBiotechnology AB, Uppsala, Sweden), followed by centrifugation tocollect lymphocytes. These lymphocytes were washed with RPMI 1640 medium(pH 7.4) supplemented with 10 v/v % fetal bovine serum and suspended ina fresh preparation of the same medium to give a cell density of 1×10⁶cells/ml. The cell suspension was distributed to a 96-well microplate ata volume of 0.15 ml/well.

A purified polypeptide obtained by the method in Example 8 was dilutedwith RPMI 1640 (pH 7.4) supplemented with 10 v/v % fetal bovine serum,and the dilution was distributed to the above microplate at a volume of0.05 ml/well. A fresh preparation of the same medium, either with orwithout 2.5 μg/ml Con A or 50 units/ml of a recombinant humaninterleukin 2 in a volume of 0.05 ml/well, was added to the microplate,and the microplate was incubated at 37° C. for 24 hours in a 5 v/v % CO₂incubator. After completion of the culture, 0.1 ml of a culturesupernatant was sampled from each well and assayed for IFN-γ activity byconventional enzyme immunosorbent assay (EIA). As a control, a systemfree of the purified protein was provided and treated similarly asabove. The results are shown in Table 3 where the IFN-γ content wasassayed and expressed in terms of international unit (IU) with respectto Gg23-901-530, an international standard for IFN-γ obtained from theNational Institutes of Health, Bethesda, Md., USA.

TABLE 3 Polypeptide IFN-γ yield (IU/ml) concentration PolypeptidePolypeptide (ng/ml) Polypeptide plus Con A plus interleukin 2 0 <0.5 <2<0.5 0.32 <0.5  6 ± 2  2 ± 1 1.6 10 ± 2  70 ± 20  60 ± 20 8 140 ± 10 490± 80 570 ± 30 40 180 ± 20 620 ± 10 880 ± 50 200 260 ± 20 800 ± 20 1500 ±400 Note: In the table, the term Polypeptide means the presentpolypeptide.

The results in Table 3 show that, lymphocytes as immunocompetent cells,produced IFN-γ by the action of the present polypeptide. It is evidentfrom the results that IFN-γ production is increased by the presence ofinterleukin 2 or Con A as a cofactor.

11-2: Increase of Cytotoxicity by NK Cell

A blood sample taken from a healthy volunteer with a heparinized syringewas diluted two-fold with PBS. The diluted blood was overlaid ontoFICOLL and centrifuged to obtain a high density layer of lymphocytes.The lymphocytes were suspended in RPMI 1640 medium (pH 7.2) containing10 μg/ml kanamycin, 5×10⁻⁵ M 2-mercaptoethanol and 10 v/v % fetal bovineserum, and the suspension was distributed to a 12-well microplate at avolume of 0.5 ml/well. A purified polypeptide obtained by the method inExample 8 was appropriately diluted with a fresh preparation of the samemedium, and the dilution was distributed to the same microplate at avolume of 1.5 ml/well, followed by adding 0.5 ml/well of a freshpreparation of the same buffer, either with or without 50 units/ml of arecombinant human interleukin 2, to the microplate, incubating themicroplate at 37° C. for 24 hours in a 5 v/v % CO₂ incubator, andwashing the resultant cells with PBS to obtain cultured lymphocytescontaining NK cells as an effector cell. 1×10⁴ cells/well aliquots ofK-562 cells (ATCC CCL243), derived from human chronic myelocyticleukemia as an NK-cell susceptive target cell, and which had beenlabelled with ⁵¹Cr in a conventional manner, were distributed to a96-well microplate, and mixed with the above NK cells in a ratio of2.5:1, 5:1 or 10:1 (effector cells:target cells). The microplate wasincubated at 37° C. for 4 hours in a 5 v/v % CO₂ incubator, followed bycounting the level of radioactivity present in each supernatant to countdead target cells. In each system, the percentage (%) of dead targetcells with respect to total target cells used in this example wascalculated to evaluate cytotoxicity. The results are shown in Table 4.

TABLE 4 Cytotoxicity Polypeptide Concentration Effector cells/concentration of interleukin 2 Target cells (pM) (Units/ml) 2.5/1 5/110/1 0 0 19 36 59 0 10 28 44 61 0.5 0 22 41 63 0.5 10 31 54 69 5 0 28 4966 5 10 36 58 71 50 0 29 53 67 50 10 42 62 72 500 0 33 56 84 500 10 5776 96 Note: In the table, the symbol (pM) means 10⁻¹² M and the termPolypeptide means the present polypeptide.

The results in Table 4 show that the polypeptide according to thepresent invention has a property of enhancing the cytotoxicity by NKcells. As evident from the results, the cytotoxicity is further enhancedby the presence of interleukin 2.

11-3: Induction of LAK Cell Formation

1×10⁴ cells/well aliquots of Raji cell (ATCC CCL86), a human Burkitt'slymphoma as an NK-cell non-susceptive target cell and which had beenlabelled with ⁵¹Cr in a conventional manner were distributed to a96-well microplate, and mixed with a cell suspension of the target cellsand cultured lymphocytes containing LAK cells as an effector cell,similarly prepared as in the method in Example 11-2 (except forculturing 72 hours), in a ratio of 5:1, 10:1 or 20:1 (effectorcells:target cells), followed by incubating the microplate at 37° C. for4 hours in a 5 v/v % CO₂ incubator and counting the level ofradioactivity present in each supernatant in a conventional manner.Thereafter, the cytotoxicity (%) was calculated in a manner similar tothat in Example 11-2. The results are shown in Table 5.

TABLE 5 Cytotoxicity Protein Concentration Effector cells/ concentrationof interleukin 2 Target cells (pM) (Units/ml) 5/1 10/1 20/1 0 0 12 23 310 10 14 25 35 0.5 0 14 24 34 0.5 10 18 32 42 5 0 16 26 37 5 10 21 36 5050 0 22 41 49 50 10 26 52 56 500 0 27 44 61 500 10 33 59 72 Note: In thetable, (pM) represents means 10⁻¹² M and the term Polypeptide means thepresent polypeptide.

The results in Table 5 show that the present polypeptide has a propertyof inducing LAK-cell formation. As evident from these results, thisinduction is further enhanced by the presence of interleukin 2.

Example 12 Acute Toxicity Test

A purified polypeptide obtained by the method in Example 8 was injectedpercutaneously, orally or intraperitioneally into 8 week-old mice in aconventional manner. The LD₅₀ of the polypeptide was found to be about 1mg/kg mouse or higher and independent of the route of administration.This demonstrates that the present polypeptide is safe to incorporateinto medicaments for administration to humans.

It is well known that IFN-γ is involved in the inhibition of bacterialinfection, in the propagation of malignant tumors, in the regulation ofhuman biophylaxis through the immunoregulatory function, and in theinhibition of immunoglobulin E antibody production. As discussed above,IFN-γ is now commercially available and is used as an agent for humandiseases susceptible to treatment with IFN-γ, and the diseases to betreated, the dose, the administration, and the safety are almostrevealed. It is reported in Cytokines in Cancer Therapy, edited byFrances R. Balkwill, translated by Yoshihiko WATANABE, published byTokyo-KagakuDojin, Tokyo, Japan (1991), that treatments using killercells such as NK- and LAK-cells are used as anti-tumor immunotherapy andapplied to human diseases, where most killer cells were reported toexert a satisfactory therapeutic effect. Recently, attention has focusedon the relationship between the therapeutic effect and the augmentationof cytotoxicity of killer cells or the induction of the formation ofkiller cells using cytokines. For example, T. Fujioka et al. reported inBritish Journal of Urology, 73:23-31 (1994) that interleukin 2 stronglyinduced the formation of LAK cells in an anti-tumor immunotherapy usingLAK cells and interleukin 2, and exerted a satisfactory effect on themetastasis of human cancer without substantially inducing serioustoxicity and side effects.

Thus, IFN-γ and killer cells are closely related with regard to thetreatment and the prevention of human diseases for complete cure andremission. With this relationship shown by the results in Examples 11and 12, the fact that the present polypeptide induces the IFN-γproduction by immunocompetent cells, enhances the cytotoxicity of NKcells, and induces the LAK cell formation indicates that apharmaceutical composition containing the present polypeptide can beadministered to humans over a relatively long period of time and canexert a satisfactory therapeutic effect on the treatment and theprevention of IFN-γ-related and/or killer cell-related diseases withoutsubstantially inducing serious side effects. Examples 13-1 to 13-8 belowdescribe preferred embodiments for the preparation of the presentpolypeptide, and Examples 14-1 to 14-6 below describe preferredembodiments of the present pharmaceutical composition for diseasessusceptible to treatment with IFN-γ.

Example 13 Preparation of Polypeptide

13-1: Preparation of Polypeptide

The immunoreaction of newborn hamsters were suppressed in a conventionalmanner by injecting a rabbit antiserum to hamster antithymus into thehamsters, where their dorsal subcutaneous tissues were transplanted withabout 5×10⁵ cells/hamster of THP-1 cells (ATCC TIB202), a myelomonocyticcell line of a human acute leukemia, and the hamsters were fed for 3weeks in a conventional manner. Tumor masses, which were about 15 gweight each and subcutaneously formed in each hamster were extracted,suspended in physiological saline in a conventional manner, and washedwith PBS.

In accordance with the method by Matthew J. Kostura et al., Proc. Natl.Acad. Sci. USA 86:5227-5231 (1989), the suspended cells were washed withten volumes of cold 20 mM HEPES buffer (pH 7.4) containing 10 mMpotassium chloride, 1.5 mM magnesium chloride, 0.1 mM EDTA, allowed tostand in 3 volumes of a fresh preparation of the same buffer for 20 minunder ice-chilled conditions, lyophilized at −80° C., and thawed todisrupt cells. The disrupted cells were centrifuged, and the supernatantwas fed to a column packed with DEAE-SEPHAROSE, (a gel for ion-exchangechromatography commercialized by Pharmacia LKB Biotechnology AB,Uppsala, Sweden) previously equilibrated with 10 mM phosphate buffer (pH6.6), followed by washing the column with 10 mM phosphate buffer (pH6.6), and eluting with a sodium chloride concentration gradient from 0 Mto 0.5 M, and collecting fractions eluting at about 0.2 M sodiumchloride.

The collected fractions were pooled, dialyzed against 10 mM phosphatebuffer (pH 6.8), fed to a column packed with DEAE 5PW (a gel forion-exchange chromatography commercialized by Tosoh Corporation, Tokyo,Japan), which had been previously equilibrated with 10 mM phosphatebuffer (pH 6.8), eluted with a linear concentration gradient of sodiumchloride from 0 M to 0.5 M in 10 mM phosphate buffer (pH 6.8), andfractions eluting at about 0.2-0.3 M sodium chloride were collected.

The resulting fractions were pooled and dialyzed against PBS. Thedialyzed solution was fed to a cylindrical plastic immunoaffinitychromatography column prepared by first packing a immunoaffinity gelcontaining a monoclonal antibody, which had been prepared according tothe method disclosed in Japanese Patent Application No.58,240/95(applied for by the present applicant), and washed with PBS. 100 mMglycine-HCl buffer (pH 2.5) was fed to the column to effectfractionation, followed by collecting eluate fractions containing apolypeptide which induces IFN-γ production by immunocompetent cells,dialyzing the fractions against sterile distilled water, concentratingthe dialyzed solution with a membrane filter, and lyophilizing theconcentrate to obtain a solid purified polypeptide. The yield was about50 ng per hamster.

13-2: Preparation of Polypeptide

Dorsal subcutaneous tissues of newborn nude mice were injected withabout 1×10⁶ cells/nude mouse of KG-1 cells (ATCC CCL246), amyelomonocytic cell line derived from human acute myelomonocyticleukemia, and the mice were fed for 4 weeks in conventional manner.Tumor masses of about 20 g weight each, which were formed subcutaneouslyin each nude mouse, were extracted and dispersed in physiological salinein a conventional manner. The cells were washed and disrupted similarlyas in Example 13-1, and the resulting mixture was purified to obtain apurified polypeptide which induces IFN-γ production by immunocompetentcells in a yield of about 20 ng per nude mouse.

The amino acid sequence of a portion of the purified polypeptide wasanalyzed in accordance with the method in Examples 9-11, revealing thatthe polypeptide has the partial amino acid sequence of SEQ ID NO:14 inthe N-terminal region and a similar molecular weight and biologicalactivity as the polypeptide in Example 8.

13-3: Preparation of Polypeptide

HL-60 cells (ATCC CCL240), a myelomonocytic cell line derived from humanacute promyelocytic leukemia, were suspended in RPMI 1640 medium (pH7.4), placed in an about 10-ml plastic cylindrical diffusion chamber inwhich a membrane filter with a diameter of 0.5 μm was installed, and thechamber was intraperitoneally embedded in an aged rat. The rat was fedfor 4 weeks in a conventional manner, and then the chamber was removed.The propagated cells in the chamber were collected, washed withphysiological saline, and disrupted similarly as in Example 13-1,followed by purifying the resulting mixture to obtain a purifiedpolypeptide which induces IFN-γ production by immunocompetent cells. Theyield was about 20 ng per rat.

The amino acid sequence of a portion of the purified polypeptide wasanalyzed in accordance with the method in Examples 9-11, revealing thatthe polypeptide has the partial amino acid sequence of SEQ ID NO:14 inthe N-terminal region and has a similar molecular weight and biologicalactivity to the polypeptide in Example 8.

13-4: Preparation of Polypeptide

THP-1 cells (ATCC TIB202), a myelomonocytic cell line derived from humanacute monocytic leukemia, were suspended in RPMI 1640 medium (pH 7.2)supplemented with 10 v/v % fetal bovine serum to give a cell density ofabout 3×10⁵ cells/ml, and cultured at 37° C. for 3 weeks in a 5 v/v %CO₂ incubator while replacing the medium with a fresh one. Thepropagated cells were separated from the resulting culture, washed withphysiological saline, and disrupted similarly as in Example 13-1,followed by purifying the resulting mixture to obtain a purifiedpolypeptide which induces IFN-γ production in a yield of about 10 ng perlitter of the culture.

The amino acid sequence of a portion of the purified polypeptide wasanalyzed in accordance with the method in Examples 9-11, revealing thatthe polypeptide has the partial amino acid sequence of SEQ ID NO:14 inthe N-terminal region and has a similar molecular weight and biologicalactivity to the polypeptide in Example 8.

13-5: Preparation of Polypeptide

Dorsal subcutaneous tissues of newborn hamsters, immunosuppressed byinjection of a rabbit antithymus serum in a conventional manner, wereinjected with about 5×10⁵ cells/head of A253 cells (ATCC HTB41), a humanepidermoid carcinoma from the submaxillary gland, and fed for 3 weeks inthe usual manner. Thereafter, tumor masses of about 10 g weight in eachhamster that were formed subcutaneously, were extracted, dispersed inphysiological saline, and washed with PBS.

The propagated cells thus obtained were washed with 20 mM HEPES buffer(pH 7.4) containing 10 mM potassium chloride, 1.5 mM magnesium chloride,and 0.1 mM EDTA, and suspended in a fresh preparation of the same bufferto give a cell density of about 2×10⁷ cells/ml. The suspended cells weredisrupted with a homogenizer, centrifuged to remove cell debris andobtain a supernatant, followed by concentrating the supernatant with anultrafiltration membrane to obtain a cell extract containing apolypeptide which induces IFN-γ production by immunocompetent cells. Theextract was purified similarly as in the method of Example 13-1,concentrated, and lyophilized to obtain a solid purified polypeptide ina yield of about 3 μg per hamster.

The purified polypeptide was sampled and analyzed in accordance with themethods in Examples 9-11, revealing that it has the amino acid sequenceof SEQ ID NO:14 in the N-terminal region and has a similar molecularweight and biological activities to those of the polypeptide in Example8.

13-6: Preparation of Polypeptide

A seed culture of A-253 cells was inoculated into RPMI 1640 medium (pH7.4) supplemented with 10 v/v % fetal calf serum and cultured in aconventional manner at 37° C. until a monolayer of cells was formed.Thereafter, the cells were detached from the surface of the culturevessel by using TRYPSIN-EDTA, a trypsin commercialized by Gibco BRL,Gaithersburg, Md., and washed with PBS. In accordance with the method inExample 13-1, the cells were disrupted, and the disrupted cells werecentrifuged to obtain a supernatant which was then incubated at 37° C.for 6 hours, purified, concentrated, and lyophilized to obtain a solidpurified polypeptide which induces IFN-γ production by immunocompetentcells in a yield of about 1 μg per 10⁷ cells. The supernatant wassampled and analyzed in accordance with the method in Example 9-11,revealing that the polypeptide has the amino acid sequence of SEQ IDNO:14 in the N-terminal region and has a similar molecular weight andbiological activities to those of the polypeptide in Example 8.

13-7: Preparation of Polypeptide

A seed culture of A-253 cells was inoculated into RPMI 1640 medium (pH7.4) supplemented with 10 v/v % fetal calf serum and cultured in aconventional manner at 37° C. until a monolayer of cells was formed.Thereafter, the culture medium was replaced with a serum-free RPMI 1640medium (pH 7.4) supplemented with 10 IU/ml of a natural IFN-γ derivedfrom KG-1 cells as an inducer, and incubated at 37° C. for 48 hours. Theculture was centrifuged to obtain a supernatant which was then purifiedby the method in Example 13-1, concentrated, and lyophilized to obtain asolid purified polypeptide which induces IFN-γ production byimmunocompetent cells in a yield of about 5 ng per 10⁷ cells.

The supernatant was sampled and analyzed in accordance with the methodin Examples 9-11, revealing that the polypeptide has the amino acidsequence of SEQ ID NO:14 in the N-terminal region and has a similarmolecular weight and biological activities to those of the polypeptidein Example 8.

13-8: Preparation of Polypeptide

A purified polypeptide obtained by the method in Example 13-1 wasdissolved in an adequate amount of sterile distilled water, and thesolution was fed to a column packed with ASAHIPAK® C4P-50 4E (a gel forhigh-performance liquid chromatography commercialized by Showa DenkoK.K., Tokyo, Japan), which had been previously equilibrated with 0.1 v/v% aqueous trifluoroacetic acid, followed by washing the column with 0.1v/v % aqueous trifluoroacetic acid and feeding to the column a lineargradient solution of acetonitrile increasing from 0 v/v % to 90 v/v % ina solution of a mixture trifluoroacetic acid and acetonitrile at a flowrate of 60 ml/hour. Fractions containing a polypeptide which inducesIFN-γ production by immunocompetent cells were collected from the elutedfractions, pooled, neutralized with 1 M aqueous Tris solution (pH 11.2),and concentrated in a conventional manner, followed by removingacetonitrile from the resulting concentrate to obtain a concentratedpolypeptide with a purity of at least 95% in a yield of about 10% byweight with respect to the material protein, d.s.b.

In accordance with the method in Example 9, the concentrated polypeptidewas sampled and analyzed for molecular weight, resulting in a singleprotein band, which induces IFN-γ production, corresponding to amolecular weight of 18,400±1,000 daltons. The amino acid sequence ofanother fresh sample was analyzed in accordance with the method inExamples 10 and 11, revealing that it has the amino acid sequence of SEQID NO:15 and SEQ ID NO:14 in the C- and N-terminal regions, and moreparticularly, the amino acid sequence of SEQ ID NO:19. Furthermore, thepolypeptide has the amino acid sequences of SEQ ID NOs:16 and 17 asinternal fragments and exhibited a similar biological activity to thepolypeptide of Example 8 even when concentrated at a relatively highlevel.

Example 14 Production Formulations

14-1: Liquid

A purified polypeptide obtained by the method in Example 13-1 wasdissolved in physiological saline containing 1 w/v % human serum albuminas a stabilizer, followed by obtaining therefrom a sterile liquidsolution. The product with a satisfactory stability can be used as aninjection, collunarium or nebula to treat and/or prevent diseasessusceptible to treatment with IFN-γ, such as malignant tumors, viraldiseases, bacterial infections, and immunopathies.

14-2: Dried Injection

A purified polypeptide obtained by the method in Example 13-2 wasdissolved in physiological saline containing 1 w/v % of a purifiedgelatin as a stabilizer, and the solution was filtered through a sterilefilter in a conventional manner. The sterile solution was distributed tovials in 1 ml volume doses, and lyophilized, followed by sealing thecaps of the vials.

The product with a satisfactory stability can be used to treat and/orprevent diseases susceptive to treatment with IFN-γ, such as malignanttumors, viral diseases, bacterial infections, and immunopathies.

14-3: Dry Injection

A solid pharmaceutical was prepared similarly as in Example 14-2 exceptthat a purified polypeptide obtained by the method in Example 13-5 andTREHAOSE® (a crystalline trehalose powder commercialized by HayashibaraCo., Ltd., Okayama, Japan), as a stabilizer were used.

The product with a satisfactory stability can be advantageously used asa dry injection for treating and/or preventing malignant tumors, viraldiseases, bacterial infections, and immunopathies.

14-4: Ointment

HI-BIS-WAKO 104, a carboxyvinylpolymer commercialized by Wako PureChemicals, Tokyo, Japan, and TREHAOSE®, a crystalline trehalose powdercommercialized by Hayashibara Co., Ltd., Okayama, Japan, were dissolvedin sterile distilled water in the amounts of 1.4 w/w % and 2.0 w/w %,respectively, and the solution was mixed to homogeneity with a purifiedpolypeptide obtained by the method in Example 13-3 and adjusted to pH7.2 to obtain a paste containing about 1 mg of a purified polypeptideper g of the paste.

The product with a satisfactory spreadability and stability can be usedto treat and/or prevent susceptive diseases such as malignant tumors,viral diseases, bacterial infections, and immunopathies.

14-5: Tablet

A purified polypeptide, obtained by the method in Example 13-4, andLUMIN(4,4′[3-[2-(1-ethyl-4-(1H)-quinolinylidene)ethylidene]propenylene]bis(1-ethylquinoliniumiodide) as a cell activator were mixed to homogeneity with FINETOSE®, ananhydrous crystalline α-maltose powder commercialized by HayashibaraCo., Ltd., Okayama, Japan, and the mixture was tableted in aconventional manner to obtain tablets of about 200 mg weight each, whichcontain the purified polypeptide and LUMIN in an amount of about 1 mgeach.

The product with a satisfactory allowability, stability andcell-activating activity can be used to treat and/or prevent susceptivediseases such as malignant tumors, viral diseases, microbism, andimmunopathies.

14-6: Agent for Adoptive Immunotherapy

Human monocytes were separated from the peripheral blood of a patientwith malignant lymphoma, and suspended in RPMI 1640 medium (pH 7.2),which had been supplemented with 10 v/v % human AB serum and preheatedat 37° C., to give a cell density of about 1×10⁶ cells/ml. The cellsuspension was mixed with about 10 ng/ml of a purified polypeptideobtained by the method in Example 13-1 and about 100 units/ml of arecombinant human interleukin 2, and incubated at 37° C. for 1 week,followed by centrifugation to collect LAK cells.

LAK cells exerted a strong cytotoxic effect on lymphoma cells whenintroduced into the patient, and the therapeutic effect is significantlyhigher than that of the conventional adoptive immunotherapy usinginterleukin 2 alone. Cytotoxic T-cells, obtained by treating a patient'stumor tissue invasive-lymphocytes instead of the patient's monocytes,showed an effect similar to LAK cells when reintroduced into thepatient. The agent for adoptive immunotherapy can be suitably applied tosolid tumors such as malignant nephroma, malignant melanoma, largeintestinal cancer, and lung cancer.

Example 15 Preparation of Polypeptide

15-1: Preparation of Transformant KGFHH2

To a 0.5-ml reaction tube were added 8 μl of 25 mM magnesium chloride,10 μl of 10×PCR buffer, 1 μl of 25 mM dNTP mix, 1 μl of 2.5 units/μl ofAmpliTaq DNA polymerase, 1 ng of a recombinant DNA containing thenucleotide sequence of SEQ ID NO:5 prepared from a phage DNA clone bythe method in Japanese Patent Application No.304,203/94 and disclosedabove in Example 5 and containing a DNA encoding the polypeptide of SEQID NO:6, and an adequate amount of a sense primer and an antisenseprimer represented by 5′-ATAGAATTCAAATGTACTTTGGCAAGCTTGAATC-3′ (SEQ IDNO:21), chemically synthesized based on an amino acid sequence near theN- and C-termini of SEQ ID NO:6, and 5′-ATAAAGCTTCTAGTCTTCGTTTTGAAC-3′(SEQ ID NO:22), and sterilized distilled water was added to give a totalvolume of 100 μl. The reaction mixture solution was successivelyincubated at 94° C. for 1 min, at 43° C. for 1 min, and at 72° C. for 1min, and this sequential incubation was repeated 3 times. The resultantmixture was further successively incubated at 94° C. for 1 min, at 6020C. for 1 min, and at 72° C. for 1 min, and this sequential incubationwas repeated 40 times to effect PCR amplification.

The resultant PCR reaction mixture and pCR-Script SK (+), a plasmidvector commercialized by Stratagene Cloning Systems, La Jolla, Calif.,USA, were ligated using DNA ligase to obtain a recombinant DNA which wasthen introduced into competent host cells of Escherichia coli XL-1 BlueMRF'Kan, a microorganism commercialized by Stratagene Cloning Systems,Calif., USA, to transform the host cells. The transformant thus obtainedwas inoculated into L-broth (pH 7.2) containing 50 μg/ml ampicillin, andcultured at 37° C. for 18 hours under shaking conditions (aeration),followed by centrifuging the resultant culture to collect theproliferated transformants and isolating recombinant DNAs using aconventional alkaline-SDS method. A part of the recombinant DNAs wasanalyzed by the dideoxy sequencing method and found to contain a DNAwhich has EcoRI and HindIII cleavage sites at the 5′- and 3′-termini ofSEQ ID NO:5, a methionine codon which initiates polypeptide synthesis,positions in the sites corresponding to those before and after the N-and C-termini of SEQ ID NO:5, and a TAG codon which terminatespolypeptide synthesis.

The remaining recombinant DNAs were cleaved with restriction enzymesEcoRI and HindIII, and 0.1 μg of the resultant EcoRI-HindIII DNAfragment obtained with DNA LIGATION KIT Version 2 (a DNA ligation kitcommercialized by Takara Shuzo Co., Ltd., Tokyo, Japan), and 10 ng ofpKK223-3 (a plasmid vector commercialized by Pharmacia LKB BiotechnologyAB Uppsala, Sweden) which had been previously cleaved with the aboverestriction enzymes, were ligated by incubating them at 16° C. for 30min to obtain a replicable recombinant pKGFHH2 DNA. By using a competentcell method, Escherichia coli Y1090 strain (ATCC 37197) was transformedwith replicable recombinant DNA pKGFHH2, and the transformant generated,KGFHH2 was inoculated into L-broth (pH 7.2) containing 50 μ/mlampicillin, and incubated at 37° C. for 18 hours under shakingconditions. The resultant culture was centrifuged to collect theproliferated transformants, a portion of which was treated by aconventional alkaline-SDS method to extract the recombinant DNA pKGFHH2.As shown in FIG. 4, the analysis by the dideoxy method revealed that, inrecombinant pKGFHH2 DNA, the KGFHH2 CDNA which contains the nucleotidesequence of SEQ ID NO:5, was ligated downstream of a Tac promoter.

15-2: Production and Purification of Polypeptide from TransformantKGFHH2

An L-broth (pH 7.2) containing 50 μg/ml of ampicillin was sterilized byautoclaving, cooled to 37° C., inoculated with the transformant KGFHH2prepared above, and incubated at the same temperature for 18 hours undershaking conditions to obtain a seed culture. Eighteen liters of a freshpreparation of the same L-broth was placed in a 20-L jar fermenter,sterilized similarly as above, cooled to 37° C., inoculated with 1 v/v %of the seed culture, and cultured at the same temperature for 8 hoursunder aeration and agitation conditions. The resultant culture wascentrifuged to collect cells, and the cells were then suspended in amixture solution (pH 7.3) consisting of 150 mM sodium chloride, 16 mMdisodium hydrogen phosphate, and 4 mM sodium dihydrogen phosphate,disrupted with ultrasound, and centrifuged to remove cell debris andobtain a supernatant.

Ammonium sulfate was added to the supernatant to give a concentration of40 w/v % and dissolved to homogeneity. The ammonium sulfate solution wascentrifuged to obtain a supernatant, which was then fed to a columnpacked with PHENYL SEPHAROSE (a product of Pharmacia LKB BiotechnologyAB, Uppsala, Sweden), which had been previously equilibrated with 10 mMphosphate buffer (pH 6.6) containing 1.5 M ammonium sulfate, followed bywashing the column with a fresh preparation of the same buffer, andfeeding a gradient buffer of ammonium sulfate ranging from 1.5 M to 0 Min 10 mM phosphate buffer (pH 6.6) to the column.

A gel for immunoaffinity chromatography was prepared and packed in aplastic cylindrical column which was then washed with PBS, fed with 10ml of fractions eluted from the above PHENYL SEPHAROSE column at about1.0 M ammonium sulfate, washed with a fresh preparation of the same PBS,and fed with 0.1 M glycine-HCl buffer (pH 2.5) containing 1 M sodiumchloride, followed by collecting fractions with IFN-γ inducibility. Thefractions were pooled, dialyzed against PBS at 4° C. overnight, andconcentrated, followed by assaying the resultant concentrate for IFN-γinducibility and protein content, which revealed that the purificationprocedure yielded about 25 mg of the polypeptide with a purity of atleast 95% per L of the culture.

Analysis further revealed that the purified polypeptide has thefollowing physicochemical properties:

(1) when electrophoresed on SDS-PAGE under non-reducing conditions, thepurified polypeptide appeared as a main polypeptide band having IFN-γinducibility at a position corresponding to 18,500±3,000 daltons;

(2) a pI of 4.9±1.0 on chromatofocusing; and

(3) the amino acid sequence containing N-terminus of the purifiedpolypeptide had the amino acid sequence of SEQ ID NO:20 which was thesame as the N-terminal sequence of SEQ ID NO:6 except that methioninewas added at the N-terminus.

Example 16 Biological Activity

16-1: Production of IFN-γ by Immunocompetent Cell

Fresh blood was collected from healthy volunteers using heparinizedsyringes, and diluted two-fold with serum-free RPMI 1640 medium (pH7.4). The diluted blood was overlaid onto FICOLL and centrifuged toobtain lymphocytes which were then washed with RPMI 1640 medium (pH 7.4)supplemented with 10 v/v % fetal calf serum, and suspended in a freshpreparation of the same medium to give a cell density of 5×10⁶ cells/ml.The cell suspension was distributed to 96-well microplates in an amountof 0.15 ml/well.

A polypeptide obtained by the method in Example 15 was diluted to givean appropriate concentration with RPMI 1640 medium (pH 7.4) supplementedwith 10 v/v % fetal calf serum. The diluted solution was distributed tothe microplates in an amount of 0.05 ml/well, followed by adding 0.05ml/well of a fresh preparation of the same medium supplemented with orwithout 2.5 μg/ml of concanavalin A or 50 units/ml of a recombinanthuman interleukin 2 to the microplates, and then incubating themicroplates at 37° C. for 24 hours in an incubator under 5 v/v % CO₂conditions. After cultivation, 0.1 ml of culture supernatant in eachwell was sampled and assayed for IFN-γ content with a conventionalenzyme immunoassay. As a control, a system free of the polypeptide wasprovided, and treated similarly as above. The results are presented inTable 6. The IFN-γ content data shown in Table 6 was calibrated usingGg23-901-530, an International Standard for Interferon, Human(HuIFN-γ),obtained from National Institutes of Health, Bethesda, Md., USA, andexpressed by international units (IU).

TABLE 6 IFN-γ productivity (IU/ml) Polypeptide Polypeptide plusPolypeptide concentration 0.5 μg/ml of 10 U/ml of (ng/ml) Polypeptideplus concanavalin A interleukin 2 0 0 0 0 1.6 1 ± 2 92 ± 32 184 ± 12 8.03 ± 1 220 ± 21  397 ± 31 40.0 6 ± 4 380 ± 34  526 ± 28 200.0 14 ± 6  549± 105 637 ± 99The results in Table 6 show that lymphocytes as immunocompetent cellproduced IFN-γ in response to the action of the polypeptide of thepresent invention. As evident from the results, the use of thepolypeptide in combination with interleukin 2 or concanavalin A as acofactor enhanced IFN-γ production.16-2: Enhancement of Cytotoxicity by NK CellFresh blood was collected from healthy volunteers using heparinizedsyringes, and diluted two-fold with 10 mM phosphate buffer (pH 7.4)containing 140 mM sodium chloride. The collected blood was overlaid ontoFICOLL, centrifuged, and then further subjected to FICOLL gradientcentrifugation to obtain high-density lymphocytes.

The lymphocytes were suspended in RPMI 1640 medium (pH 7.2) containing10 μg/ml kanamycin, 5×10⁻⁵ M 2-mercaptoethanol, and 10 v/v % fetal calfserum to give a cell density of 1×10⁶ cells/ml. The cell suspension wasdistributed into 12-well microplates in an amount of 0.5 ml/well. Apolypeptide obtained by the method in Example 15 was appropriatelydiluted with a fresh preparation of the same medium, and the dilutedsolution was distributed to the microplates in an amount of 1.5 ml/well,followed by distributing 0.5 ml/well of a fresh preparation of the samemedium with or without 50 units/ml of a recombinant human interleukin 2.The microplates were incubated in an incubator at 37° C. for 24 hoursunder 5 v/v % CO₂ conditions, followed by washing with 10 mM phosphatebuffer (pH 7.4) containing 140 mM sodium chloride to obtain culturedlymphocytes containing NK cells as effector cells. K-562 cells (ATCC CCL243), derived from human chronic myelogenous leukemia, as NKcell-sensitive target cells labelled in the usual manner with ⁵¹Cr, weredistributed to 96-well microplates to give 1×10⁴ cells/well. Effectorcells were added to each well in the ratio (effector cells):(targetcells) of 2.5:1, 5:1 or 10:1, and incubated in an incubator at 37° C.for 4 hours under 5 v/v % CO₂ conditions. According to what isconventionally done, the radioactivity of each supernatant in each wellwas measured to count the dead target cells. In each system, thepercentage (%) of dead target cells to target cells was calculated todetermine the cytotoxicity level. The results are presented in Table 7.

TABLE 7 Cytotoxicity (%) (Effector Concentration of Concentration ofcell):(Target cell) polypeptide (pM) interleukin 2 (unit/ml) 2.5:1 5:110:1 0 0 22 35 65 0 10 30 48 73 0.5 0 23 36 66 0.5 10 32 50 75 5 0 25 3968 5 10 35 52 78 50 0 29 47 73 50 10 41 59 85 500 0 37 50 83 500 10 5270 93 Note: In the Table, (pM) means 10⁻¹² M.

The results in Table 7 show that the polypeptide has an activity ofenhancing the cytotoxicity by NK cells. As also shown in Table 7, thepresence of interleukin 2 further enhances cytotoxicity.

16-3: Induction of LAK Cell Formation

According to what is done conventionally, ⁵¹Cr-labelled Raji cells (ATCCCCL 86), derived from human Burkitt lymphoma as a target cell resistantto NK cells, were placed in 96-well microplates to give 1×10⁴cells/well, and cultured for 72 hours. Cultured lymphocytes, containingLAK cells as effector cells prepared similarly as above, and targetcells were added to the microplates in the ratio of 5:1, 10:1 or 20:1,and the microplates were incubated in an incubator at 37° C. for 4 hoursunder 5 v/v % CO₂ conditions. Thereafter, the radioactivity of eachsupernatant in each well was measured, and the cytotoxicity (%) wascalculated similarly as above. The results are presented in Table 8.

TABLE 8 Cytotoxicity (%) (Effector Concentration of Concentration ofcell):(Target cell) polypeptide (pM) interleukin 2 (unit/ml) 5:1 10:120:1 0 0 11 21 34 0 10 15 28 38 0.5 0 13 22 35 0.5 10 17 31 43 5 0 15 2339 5 10 19 34 48 50 0 20 25 44 50 10 23 42 54 500 0 27 34 57 500 10 3154 67 Note: In the Table, (pM) means 10⁻¹² M.

The results in Table 8 show that the present polypeptide has an activityof inducing LAK cells. As is shown in the results, the presence ofinterleukin 2 further enhances the induction.

Example 17 Acute Toxicity Test

According to what is done conventionally, a purified polypeptideobtained by the method in Example 15 was percutaneously, perorally orintraperitoneally administered to 8-week-old mice. As a result, the LD₅₀of the purified polypeptide was about-1 mg/kg or higher, independent ofthe route of administration. This provides evidence that the polypeptideaccording to the present invention can be safely incorporated intopharmaceuticals for administration to humans.

Example 18 Production Formulations

18-1: Solution

A polypeptide obtained by the method in Example 15, was dissolved inphysiological saline containing 1 w/v % human serum albumin as astabilizer to obtain a 1 mg/ml polypeptide solution which was thensterilized by membrane filter to obtain a solution. The resultantproduct with a satisfactory stability can be used in the treatmentand/or the prevention of diseases susceptible to treatment with IFN-γ,such as malignant tumors, viral diseases, bacterial infectious diseases,and immune diseases.

18-2: Dry Injection

A polypeptide obtained by the method in Example 15, was dissolved in 100ml physiological saline containing 1 w/v % purified gelatin as astabilizer, and the solution was sterilized with a membrane filter inthe usual manner. One ml aliquot doses of the sterilized solution weredistributed to vials, lyophilized, and cap sealed. The product with asatisfactory stability can be used as a dry injection for treatingand/or preventing diseases susceptible to treatment with IFN-γ, such asmalignant tumors, viral diseases, bacterial diseases, and immunediseases.

18-3: Ointment

HI-BIS-WAKO 104, a carboxyvinylpolymer commercialized by Wako PureChemicals, Tokyo, Japan, and a purified trehalose were dissolved indistilled water to give concentrations of 1.4 w/w % and 2.0 w/w %,respectively, in a solution. A polypeptide obtained by the method inExample 15 was then dissolved to homogeneity in the solution, followedby adjusting the pH of the resultant solution to pH 7.2 to obtain apaste containing about 1 mg/g of the polypeptide. The product with asatisfactory spreadability and stability can be used as an ointment fortreating and/or preventing diseases susceptible to treatment with IFN-γ,such as malignant tumors, viral diseases, bacterial infectious diseases,and immune diseases.

18-4: Tablet

A polypeptide obtained by the method in Example 15, and LUMIN, i.e.,[4,4′-[3-[2-(1-ethyl-4(1H)-quinolinylidene)ethylidene]propenylene]bis(1-ethylquinoliniumiodide, as a cell activator were mixed to homogeneity with FINETOSE®, ananhydrous crystalline α-maltose commercialized by Hayashibara Co., Ltd.,Okayama, Japan, and the mixture was tableted in the usual manner by atableting machine to obtain tablets of about 200 mg weight each whichcontain the polypeptide and LUMIN at about 1 mg each. The product havinga satisfactory swallowing ability, stability, and cell activatingactivity, can be used as a tablet for treating and/or preventingdiseases susceptive to treatment with IFN-γ, such as malignant tumors,viral diseases, bacterial infectious diseases, and immune diseases.

Example 19 Adoptive Immunotherapeutic Agent

Mononuclear cells were isolated from the peripheral blood of a patientwith malignant lymphoma, suspended in a RPMI 1640 medium (pH 7.2)supplemented with 10 v/v % human AB serum, and preheated to 37° C. togive a cell density of about 1×10⁶ cells/ml. The cells were mixed withabout 1.0 μg/ml of a polypeptide obtained by the method in Example 15,and about 100 units/ml of a recombinant human interleukin 2, followed byincubating in a 5 v/v % CO₂ incubator at 37° C. for 1 week, andcentrifuging the resultant culture to collect LAK cells.

The LAK cells thus obtained exhibited a strong cytotoxicity on lymphomacells when introduced into the donor patient, and exerted a highercytotoxicity than that attained by the adoptive immunotherapy usinginterleukin 2 alone. Cytotoxic T-cells obtained by similarly treatinglymphocytes which invaded into the tumor tissues of the patient, wereinjected into the donor patient in place of the above lymphocytes andresulted in exertion of a similar effect as in LAK cells. The adoptiveimmunotherapeutic agent can be arbitrarily used to treat solid malignanttumors, such as renal cancer, malignant melanoma, colonic cancer, rectalcancer, and lung cancer.

Example 20 Preparation of Hybridoma H-1

Transformant KGFHH2 was generated as described in Example 15.Polypeptides produced in culture from this transformant were centrifugedin ammonium sulfate and the supernatant was fractionated by elution on aPHENYL SEPHAROSE column as described in Example 15.

Fractions eluted at around 1.0 M ammonium sulfate from the PHENYLSEPHAROSE column were pooled, membrane filtered, dialyzed against 10 mMphosphate buffer (pH 6.5) at 4° C. for 18 hours, and fed to a columnpacked with DEAE 5PW (a product commercialized by Tosoh Corporation,Tokyo, Japan), which had been previously equilibrated with 10 mMphosphate buffer (pH 6.5), followed by washing the column with a freshpreparation of the same buffer, and feeding a linear gradient buffer ofsodium chloride ranging from 0 M to 0.2 M in 10 mM phosphate buffer (pH6.5) to the column while collecting fractions eluting at 0.05 M sodiumchloride.

Thereafter, the fractions were concentrated with a membrane and fed to acolumn packed with SUPERDEX 75 (a product of Pharmacia LKB biotechnologyAB, Uppsala, Sweden), which had been equilibrated with phosphatebuffered saline, followed by feeding a fresh preparation of PBS to thecolumn to collect fractions corresponding to about 18,500 daltons. Anaqueous solution containing about 5.2 mg of a purified protein wasobtained with the total yield throughout the purification being about10%.

Analysis according to Example 15 revealed that the purified polypeptideappeared as a main protein band having an IFN-γ inducibility at aposition corresponding to 18,500±3,000 daltons when electrophoresed inSDS-PAGE under reducing conditions, and had a pI of 4.9±1.0 on chromatofocusing. The amino acid sequence containing the N-terminus of thepurified protein was also determined to have the amino acid sequence ofSEQ ID NO:20, which corresponds to the N-terminal sequence of SEQ IDNO:6 with a methionine added to its N-terminus.

Ten-week-old BALB/c mice were each intraperitoneally injected with 20 μgof a purified polypeptide, obtained above from the transformant KGFHH2in combination with a complete Freund's adjuvant. The mice were furtherintraperitoneally injected twice with the same dose at an interval oftwo weeks with an intravenous injection of the same dose 1 week afterthe final intraperitoneal injection. The splenocytes were prepared fromthe mice and suspended to obtain a cell suspension.

The spleen cells and SP2/O-Ag14 cells from mouse myeloma cells (ATCC CRL1581) were suspended in RPMI 1640 medium (pH 7.2) preheated to 37° C. atcell densities of 3×10⁴ cells/ml and 1×10⁴ cells/ml, respectively, andcentrifuged to collect sediment. One ml of a serum-free RPMI 1640 medium(pH 7.2) containing 50 w/v % polyethylene glycol with an averagemolecular weight of 1,500 daltons was added dropwise to the precipitateover 1 min. The mixture was incubated at 37° C. for 1 min, followed byadding a serum-free RPMI 1640 medium (pH 7.2) dropwise to the mixture tobring the volume up to 50 ml, centrifuging the mixture, and collectingthe sediment formed. The sediment thus obtained was suspended in HATmedium, hdistributed to 96-well microplates in an amount of 200 μl/well,and incubated at 37° C., for 1 week, followed by selecting forhybridomas.

The amount of antibodies secreted in the supernatant in each well wasassayed on enzyme immunoassay based on the immunoreaction of theantibodies and purified polypeptide obtained above from the transformantKGFHH2. Hybridomas capable of producing antibodies which stronglyreacted with the purified polypeptide were selected. A cloned hybridomaH-1 cell capable of producing the present monoclonal antibody wasobtained in the usual manner by repeatedly treating these hybridomaswith limiting dilution.

Example 21 Preparation of Monoclonal Antibody H-1mAb and its Analysis onWestern Blot Technique

21-1: Preparation of Monoclonal Antibody H-1mAb

Hybridoma H-1 cells obtained in Example 20 were suspended in RPMI 1640medium (pH 7.2) supplemented with 5 v/v % calf serum to give a celldensity of about 1×10⁶ cells/ml, and incubated in an incubator at 37°C., under 5 v/v % CO₂ conditions while scaling up the culture. When thecell density of the culture reached a prescribed level, 1×10⁷cells/mouse of the proliferated hybridoma H-1 cells wereintraperitoneally injected into 8 week old BALB/c mice, which had beenintraperitoneally injected previously with 0.5 ml/mouse of pristane,followed by feeding the mice in the usual manner for one week.

Ascites were collected from the mice, diluted three-fold with PBS, mixedwith ammonium sulfate to give a saturation degree of 50 w/v %, allowedto stand at 4° C. for 24 hours, and centrifuged to collect sediment. Thecollected sediment was dialyzed against an aqueous solution of 20 mMpotassium dihydrogen phosphate (pH 6.7) at 4° C. overnight, and fed to acolumn of hydroxyapatite which had been previously equilibrated with afresh preparation of the same aqueous solution, followed by feeding tothe column a linear gradient of a potassium dihydrogen phosphate buffer(pH 6.7) ranging from 20 mM to 300 mM to obtain an aqueous solutioncontaining the present monoclonal antibody H-1mAb. The yield was about 5mg per mouse. Conventional analysis revealed that the antibody belongsto the IgG₁ class.

21-2: Analysis on Western Blot Technique

One μg of the purified polypeptide obtained in Example 15-2 was added toa solution containing 100 mg dithiothreitol, 0.5 ml of an aqueoussolution of 10 w/v % SDS, and 1 ml of glycerol, and the mixture wasincubated at 37° C. for 1 hour and electrophoresed on SDS-PAGE. Thepolypeptide on the gel was transferred to a nitrocellulose membrane inthe usual manner and the nitrocellulose membrane was then soaked in aculture supernatant of hybridoma H-1 cells for 1 hour, and washed with50 mM Tris-HCl buffer (pH 7.5) containing 0.05 v/v % TWEEN 20 to removeexcessive amounts of antibodies. The membrane was further soaked for 1hour in PBS containing an anti-mouse Ig antibody prepared from rabbitsto effect an immunoreaction, washed with 50 mM Tris-HCl buffer (pH 7.5)containing 0.05 v/v % TWEEN 20, and soaked in 50 mM Tris-HCl buffer (pH7.5) containing 0.005 v/v % hydrogen peroxide and 0.3 mg/ml3,3′-diaminobenzidine to provide a change in color.

As a control, a system using a recombinant human interleukin 12 in placeof the purified polypeptide was provided, and treated similar to thepurified polypeptide above. Calf serum albumin (MW=67,000 daltons),ovalbumin (MW=45,000 daltons), carbonic anhydrase (MW=30,000 daltons),trypsin inhibitor (MW=20,100 daltons), and α-lactalbumin (MW=14,400daltons) were used as marker proteins. The results are shown in FIG. 5.

As is evident from FIG. 5, the monoclonal antibody H-1mAb specificallyreacted with the purified polypeptide (lane 1) obtained in Example 15-2,but did not react with human interleukin 12 (lane 2). This demonstratesthat the present monoclonal antibody reacts specifically and notnon-specifically to the polypeptide obtained in Example 15-2.

Example 22 Preparation of Hybridoma H-2 and Monoclonal Antibody H-2mAb

Hybridoma H-2, which produces a monoclonal antibody H-2, was preparedsimilarly as in Example 21, except that P3-X63-Ag8 cells (ATCC TIB9)were used in place of SP/0-14Ag cells. Hybridoma H-2 was proliferatedsimilarly as in Example 21, and about 5.6 mg of monoclonal antibodyH-2mAb per BALB/c mouse was purified from the ascites. Conventionalanalysis revealed that the monoclonal antibody belongs to the IgM class,and it specifically reacted with the polypeptide purified in Example15-2 when analyzed on Western blots as similarly done in Example 21.

Example 23 Purification of Polypeptide on Immunoaffinity Chromatography

Eighty mg of monoclonal antibody H-1mAb obtained in Example 21-1 wasweighed and dialyzed against 0.1 M borate buffer (pH 8.5) containing 0.5M sodium chloride at 4° C. overnight. Four g of CNBr-activated SEPHAROSE4B (a water-insoluble carrier commercialized by Pharmacia LKBBiotechnology AB, Uppsala, Sweden) was swelled with 1 mM of an aqueouschloric acid solution, successively washed with a fresh preparation ofthe same buffer and 0.1 M borate buffer (pH 8.5) containing 0.5 M sodiumchloride, admixed with about 10 ml of the aqueous monoclonal antibodysolution obtained above, and successively incubated at ambienttemperature and at 4° C. overnight under gentle stirring conditions.Thereafter, the resultant gel was successively washed with 1 M aqueousethanolamine solution (pH 8.0), 0.1 M borate buffer (pH 8.5) containing0.5 M sodium chloride, and 0.1 M acetate buffer (pH 4.0), and thesewashing steps were repeated five times. Finally, the gel was washed withPBS to provide a gel for immunoaffinity chromatography. Conventionalanalysis revealed that about 6 mg monoclonal antibody H-1mAb was linkedto 1 ml of the gel.

Ten ml of the gel for immunoaffinity chromatography was packed in aplastic cylindrical.column, washed with PBS, and fed with 10 ml of aPHENYL SEPHAROSE eluted fraction containing about 0.1 mg/ml of thepolypeptide purified in Example 15-2. The column was washed with a freshpreparation of PBS, and fed with 0.1 M glycine-HCl buffer (pH 2.5)containing 1 M sodium chloride to collect fractions with an IFN-γinducing activity. The fractions were pooled, dialyzed against PBS at 4°C. overnight, concentrated and assayed the IFN-γ inducing activity andprotein content, revealing that this purification procedure yielded apurified polypeptide with a purity of 95 w/w % or higher in a yield ofabout 100%.

Example 24 Detection of Polypeptide on Enzyme Immunoassay

Rabbits were immunized in the usual manner with a purified polypeptideobtained in Example 15-2, and their blood was later collected.Immunoglobulin G antibody was isolated from the blood, dissolved in PBSto give a concentration of 20 μg/ml, and the solution was distributedinto 96-well microplates in an amount of 100 μl/well. The microplateswere incubated at ambient temperature for 3 hours, followed by removingsolutions containing IgG from the microplates, adding PBS containing 1w/v % calf serum albumin to the microplates in an amount of 200 μl/well,and allowing them to stand at 4° C. overnight.

Phosphate buffered saline was removed from the microplates and themicroplates were then washed with PBS containing 0.05 v/v % TWEEN 20,and poured with 100 μl/well of a solution prepared by appropriatelydiluting the polypeptide with PBS containing 0.5 w/v % calf serumalbumin, followed by reacting the mixture solution at ambienttemperature for 2 hours under shaking conditions. The microplates werewashed with PBS containing 0.05 v/v % TWEEN 20, and 100 μl/well of asolution containing a monoclonal antibody H-1mAb labelled with biotinwas added, followed by reacting the solution at ambient temperature for2 hours under shaking conditions, washing the microplates with PBScontaining 0.05 v/v % TWEEN 20, adding 100 μl/well of a solutioncontaining a complex of horseradish peroxidase and streptoavidin, andfurther reacting the resultant mixture at ambient temperature for 2hours under shaking conditions. The microplates were then washed withPBS containing 0.05 v/v % TWEEN 20, and the activity of the horseradishperoxidase linked to the purified polypeptide was measured forabsorbance at a wavelength of 492 nm using o-phenylenediamine assubstrate. The results are shown in Table 9.

TABLE 9 Concentration of Absorbance Relative error polypeptide (pg/ml)at 492 nm* (%) 1,000 1.51 ± 0.05 3.3 500 0.93 ± 0.05 5.4 250 0.55 ± 0.035.5 100 0.25 ± 0.02 8.0 50 0.137 ± 0.007 5.1 25 0.080 ± 0.007 8.8 00.024 ± 0.007 — Note: The symbol * means a statistical value of triplet.

As evident from the results in Table 9, the detection method accordingto the present invention accurately assays the polypeptide in an amountin the range of about 50-1,000 pg/ml.

Example 25 Detection of Polypeptide on Radioimmunoassay

Rabbits were immunized with the polypeptide purified in Example 15-2 inthe usual manner, and their blood was later collected, followed byisolating an IgG antibody. The antibody was adsorbed onto polystyrenebeads for radioimmunoassay in the usual manner, and allowed to stand inPBS containing 2 w/v % calf serum albumin at 4° C. overnight to obtainan immobilized antibody.

One polystyrene bead with the antibody adsorbed thereto was placed in atest tube, soaked in 0.2 ml of a solution prepared by diluting thepurified polypeptide of Example 20 with PBS containing 0.5 w/v % calfserum albumin, and allowed to stand at 4° C. for 4 hours. The bead wasthen washed with PBS containing 0.05 v/v % TWEEN 20 and 0.5 w/v % calfserum albumin, soaked in 0.2 ml (1×10⁵ cpm) of a solution containingmonoclonal antibody H-2mAb obtained in Example 22, labelled with ¹²⁵I inthe usual manner, and allowed to stand at 4° C. overnight. Afterremoving an excessive amount of ¹²⁵I-labelled antibody, the bead waswashed with PBS containing 0.05 v/v % TWEEN 20 and 0.5 w/v % calf serumalbumin, followed by counting the radioactivity of the bead on agamma-counter. The results are shown in Table 10.

TABLE 10 Concentration of Count* Relative error polypeptide (pg/ml)(cpm) (%) 1,000.0 6,900 ± 200 2.9 500.0 4,100 ± 20 0.5 250.0 2,390 ± 502.1 125.0 1,590 ± 70 4.4 62.5   880 ± 10 1.1 0   700 ± 20 — Note: Thesymbol * means a statistical value of triplet.

As evident from the results in Table 10, the present detection methodaccurately assays for the polypeptide in the range of about 100-1,000pg/ml.

While there has been described what is at present considered to be thepreferred embodiments of the invention, it will be understood thevarious modifications may be made therein, and it is intended to coverin the appended claims all such modifications as fall within the truespirit and scope of the invention.

1. A monoclonal antibody which binds to the polypeptide of SEQ ID NO:8.